COX-2 function and wound healing

ABSTRACT

The invention relates to compositions and methods for enhancing bone healing, bone formation and wound healing. More specifically, it relates to the use of cyclooxygenase 2 (COX-2) following bone fracture, orthopaedic procedure or wound infliction to enhance healing.

FIELD OF THE INVENTION

[0001] This invention relates to the field of cyclooxygenase activityand wound healing.

BACKGROUND OF THE INVENTION

[0002] Bones, along with a small number of cartilages, comprise theskeletal system that serves as the rigid supporting framework of thebody in adult humans. Certain parts of the supporting framework formchambers, such as the skull and the thoracic cage, that are importantfor protecting the soft parts contained in the chambers. Bones alsoserve as attachments for muscles and act as levers in the joint systemof the body.

[0003] Mature bone is comprised of an organic framework of fibroustissue and inorganic salts known as crystalline hydroxyapatite (HA). HAis composed of calcium and phosphorous, which are derived from the bloodplasma and ultimately from nutritional sources. HA represents about 60percent of the weight of compact bone and is deposited on a fibrousstructure of collagenous connective tissue. Without HA, bone loses mostits weight and rigidity and is susceptible to damage.

[0004] The process of bone formation, also known as osteogenesis,involves three main steps: production of the extracellular organicmatrix (osteoid); mineralization of the matrix to form bone; and boneremodeling by resorption and reformation. The cellular activities ofosteoblasts, osteocytes, and osteoclasts are essential to the process.

[0005] Osteoblasts synthesize the collagenous precursors of bone matrixand also regulate its mineralization. During bone formation, osteoblastsline tiny spaces known as lacunae within the surrounding mineralizedmatrix. Osteoblasts that line the lacunae are called osteocytes.Osteocytes occupy minute canals (canaliculi) which permit thecirculation of tissue fluids. Hormones, growth factors, physicalactivity, and other stimuli act mainly through osteoblasts to bringabout effects on bone. Osteoclasts are derived from hematopoietic stemcells that also give rise to monocytes and macrophages. Osteoclastsadhere to the surface of bone undergoing resorption and lie indepressions referred to as resorption bays. Osteoclasts are apparentlyactivated by signals from osteoblasts. Osteoclastic bone resorption doesnot occur in the absence of osteoblasts. To meet the requirements ofskeletal growth and mechanical function, bone undergoes dynamicremodeling by a coupled process of bone resorption by osteoclasts andreformation by osteoblasts.

[0006] Bone is formed through one of two pathways, by replacement ofcartilage or by direct elaboration from periosteum. These processes areknown, respectively, as endochondral ossification and intramembranousossification. During endochondral ossification, a cartilaginous bonemodel is first formed. Then, a layer of bone on the surface of thecartilaginous shaft is formed by osteoblasts. Succeeding layers of bonefollow. At the same time, the matrix of cartilage cells is calcifiedinto a trabecular network of cartilage while the interstitial cartilageis degenerated. The combined processes of calcification and degenerationof the cartilage advance from the center toward the ends of thecartilage model. The osteoblasts penetrate the cartilage along withcapillaries to produce bone on the cartilaginous trabeculae and advancefrom the center to the ends to progressively form bone on thecartilaginous trabeculae. Ultimately, the calcified cartilage iscompletely replaced by spongy bone.

[0007] In contrast, the process of intramembranous ossification does notinvolve a cartilaginous template. Instead, mesenchymal cells becomeosteoblasts which begin to form the branching trabeculae of bone. Theinitial thin trabeculae are some times referred to as spicules. Thetrabecular bone becomes denser by widening of the trabeculae, and isthen remodeled externally and internally. The mandibles, clavicles andcertain bones of the skull are produced through intramembranousossification.

[0008] There are a number of diseases related to bone formation,deterioration and healing, including osteoporosis, osteogenesisimperfecta (OI) and fibrodysplasia ossificans progressiva (FOP).Osteoporosis, or porous bone, is a disease characterized by low bonemass and structural deterioration of bone tissue, leading to bonefragility and an increased susceptibility to fractures of the hip,spine, and wrist. Osteoporosis is a major public health threat for morethan 28 million Americans, 80 percent of whom are women. The strength ofbone depends on its mass and density. Bone density depends in part onthe amount of calcium, phosphorus and other minerals bones contain.Bones that contain less mineral are weakened and lose internalsupporting structure. A full cycle of bone remodeling takes about 2 to 3months. Children tend to make new bone faster than old bone is brokendown. As a result, bone mass increases. Peak bone mass is reached in anindividual's mid-30s. Although bone remodeling continues, old bone isbroken down faster than new bone is formed. As a result, adults loseslightly more bone than is gained—about 0.3 percent to 0.5 percent ayear. Lack of vitamin D and calcium in an individual's diet canaccelerate the process. In addition, for women at menopause, estrogenlevels drop and bone loss accelerates to about 1 percent to 3 percent ayear. Bone loss slows but doesn't stop at around age 60. Women may losebetween 35 percent and 50 percent of their bone mass, while men may lose20 percent to 35 percent of their bone mass. Development of osteoporosisdepends on the bone mass attained between ages 25 and 35 (peak bonemass) and how rapidly it is lost as an individual get older. The higheran individual's peak bone mass, the less likely that individual willdevelop osteoporosis. Calcium, vitamin D and exercising regularly areimportant for maintaining bone strength. Nonetheless, methods foreffectively treating osteoporosis are still desired.

[0009] Osteogenesis Imperfecta (OI) is a genetic disorder characterizedby bones that break easily, often from little or no apparent cause.There are at least four distinct forms of the disorder, representingextreme variation in severity from one individual to another. Forexample, a person may have as few as ten or as many as several hundredfractures in a lifetime. While the number of persons affected with OI inthe United States is unknown, the best estimate suggests a minimum of20,000 and possibly as many as 50,000. OI can be dominantly orrecessively inherited and can also occur as a mutation. A cure for OIhas not yet been discovered. As a result, methods for treatment focus onpreventing and controlling symptoms, strengthening bone mass andensuring proper healing.

[0010] In addition, both osteoporosis and OI leave patients vulnerableto bone fractures. If these bone fractures do not heal properly, thesepatients may continue to suffer from pain and may be at increased riskfor further fractures as well as other related complications. Methods ortreatments that enhance and/or ensure proper fracture healing areimportant for patients with osteoporosis or brittle bone disease.Another group that will benefit from methods for enhanced wound healingare the elderly and patients who have undergone orthopaedic procedures.

[0011] Fracture healing is the culmination of a highly orchestratedseries of physiological and cellular pathways to restore the function ofbroken bones. Fracture healing generally involves the following steps:the formation of a hematoma (collection of blood at the fracture site),development of a soft callus due to cell multiplication in the lining ofthe injured bone, growth of blood vessels and fibrocartilege in themiddle of the fracture, formation of osteoblasts that migrate into thecallus and deposit calcium to form a hard callus, and remodeling andstrengthening of the bone through osteoblast and osteoclast formation.

[0012] Osteogenesis during fracture healing occurs by intramembraneousand endochondral ossification that histologically resembles fetalskeletogenesis (Einhorn 1998; Vortkamp et al. 1998; Ferguson et al.1999). However, the localized tissue hypoxia, the fracture hematoma,subsequent inflammation at the fracture site, and the frank remodelingof the fracture callus at the later stages of healing are uniquephysiological and cellular responses to bone fractures that have noknown corresponding counterpart during fetal development of theskeleton.

[0013] It has been hypothesized that the early physiological responsesto a bone fracture, namely hypoxia and inflammation, induce geneexpression pathways and promote cell proliferation and migration intothe fracture site in order to promote healing (Brighton et al. 1991;Bolander 1992). Production or release of specific growth factors,cytokines, and local hormones at the fracture site by thesephysiological processes would create the appropriate microenvironment to(1) stimulate periosteal osteoblast proliferation and intramembraneousossification to form the hard fracture callus, (2) stimulate cellproliferation and migration into the fracture site to form the softcallus, and (3) stimulate chondrocyte differentiation in the soft calluswith subsequent endochondral ossification. Remodeling of the fracturecallus by osteoclastic resorption and subsequent osteogenesis convertsthe fracture callus woven bone into cortical bone and thereby restoresthe shape and mechanical integrity of the fractured bone.

[0014] One potential class of factors that would mediate certain eventsof fracture healing is the prostaglandins. The effects of prostaglandinson bone metabolism are complex since prostaglandins can stimulate boneformation as well as bone resorption (Kawaguchi et al. 1995). However,because the in vivo half-life of purified or synthetic prostaglandins isvery short, prostaglandins per se have a limited therapeutic value.

[0015] Prostaglandins are synthesized by osteoblasts and different cellstimuli can alter the amount and possibly the spectrum of prostaglandinsproduced by osteoblasts (Feyen et al. 1984; Klein-Nulend et al. 1997;Wadleigh and Herschman 1999). Therefore, signal transduction, mechanicalperturbations, or other physiological signals can affect bone metabolismthrough altercation of prostaglandin production.

[0016] Prostaglandin synthesis begins with the release of arachidonicacid from membrane phospholipids by phospholipase activity. Arachidonicacid is subsequently converted into prostaglandin H₂ (PGH₂) bycyclooxygenase (COX) via two independent catalytic steps (Needleman etal. 1986). Synthase enzymes then convert PGH₂ into the specificprostaglandins produced by that cell such as PGD₂, PGE₂, PGF_(2α),prostacyclin, and thromboxane. Thus, cyclooxygenase activity isessential for normal prostaglandin production and cyclooxygenase isbelieved to be the rate-limiting enzyme in the prostaglandin syntheticpathway.

[0017] There are two known forms of cyclooxygenase, COX-1 and COX-2,which are encoded by two genes (Xie et al. 1991; O'Banion et al. 1992).COX-1 is constitutively expressed by many tissues and provides ahomeostatic level of prostaglandins for the body and specific organs,such as the stomach and kidneys (Vane et al. 1998). In contrast, COX-2is inductively expressed in vitro by a diverse array of cell stimulisuch as exposure to lipopolysaccharide (O'Sullivan et al. 1992a;O'Sullivan et al. 1992b), certain cytokines and growth factors (O'Banionet al. 1992; Wadleigh and Herschman 1999), or mechanical stress (Topperet al. 1996; Klein-Nulend et al. 1997). COX-2 expression can bestimulated in vivo by wounding and inflammation (Masferrer et al. 1994;Shigeta et al. 1998; Muscaráet al. 2000).

[0018] Inhibiting the cyclooxygenase activity of COX-1 and COX-2 canreduce prostaglandin synthesis by preventing the conversion ofarachidonic acid into PGG₂, the precursor of PGH₂. This is commonly doneto reduce inflammation and pain with aspirin and non-steroidalanti-inflammatory drugs (NSAIDs), such as indomethacin. Most NSAIDsinhibit the cyclooxygenase activity of COX-1 and COX-2 with near equalpotency, which often leads to detrimental gastro-intestinal or kidneyside effects (Raskin 1999; Whelton 1999). Use of COX-2-selective NSAIDshas become very popular since these drugs, such as celecoxib (Celebrex)and rofecoxib (Vioxx) preferentially inhibit the cyclooxygenase activityof COX-2 with selectivity relative to COX-1 of approximately 8-fold forcelecoxib and 35-fold for rofecoxib (Riendeau et al. 2001).

[0019] Prostaglandins are produced during fracture healing.Prostaglandin levels in and around the healing callus of rabbit tibiathat had been severed by osteotomy showed that PGE and PGF levels wereelevated between 1 and 14 and 7 and 14 days post-osteotomy, respectively(Dekel et al. 1981). No survey of the temporal pattern or variety ofprostaglandins produced during fracture healing has been reported forother rodents or man. Non-specific NSAIDs have been shown to delay butnot stop fracture healing in experimental animal models (Rø et al. 1976;Allen et al. 1980; Altman et al. 1995). In addition, non-specific NSAIDshave been shown to reduce the incidence and severity of heterotopic(abnormal or deviating from the natural position) bone formation inhumans following certain fractures or orthopaedic surgical procedures(Pritchett 1995; Moore et al. 1998). These observation suggest thatprostaglandins are necessary for bone formation but given thelimitations of non-specific NSAID use, it is unknown whetherprostaglandins produced by COX-1, COX-2 or both enzymes are essentialfor fracture healing.

SUMMARY OF THE INVENTION

[0020] The present invention relates to compositions and methods for usein wound healing and for use in enhancing fracture healing, boneformation and wound healing. The present invention further provides formethods for treating diseases related to bones, including osteoporosis,osteogenesis imperfecta and fibrodysplasia ossificans progressiva.

[0021] One embodiment of the present invention involves a vector for usein wound healing comprising a promoter linked to a cyclooxygenaseexpression cassette. In a further embodiment, the vectors of thisinvention may be used in gene therapy approaches to enhance woundhealing. The wound conditions of this invention can include, bonefractures and skin lesions. The methods of this invention areparticularly useful for wound healing in the elderly, patients withosteoporosis and OI, and patients that suffer from delayed woundhealing.

[0022] In another embodiment, cyclooxgenase proteins, including COX-1,COX-2 or a combination of the two, are formulated as pharmaceuticalcompositions for use in wound healing. In a further embodiment of theinvention, the pharmaceutical compositions are combined with a carrierfor applications in wound healing.

[0023] Another embodiment of the invention provides for the use of thevectors of this invention in gene therapy approaches and/or thepharmaceutical compositions to treat osteoporosis, OI and other relatedbrittle bone conditions. In a further embodiment, the vectors andcompositions are used therapeutically to counteract conditionsassociated with osteoporosis, OI and brittle bones conditions. In afurther embodiment, the vectors and compositions are used for woundhealing and/or to enhance wound healing in patients with osteoporosis,OI or brittle bone conditions.

[0024] In another embodiment of the invention, COX-2 selective NSAIDsare used in the treatment of heterotopic ossification conditions. Suchconditions can include fibrodysplasia ossificans progressiva. Inaddition, heterotopic ossification can occur following hip replacementprocedures and after acetabular fractures. COX-2 selective NSAIDs can beused to treat heterotopic ossification under these circumstances aswell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1. Radiographic Analysis of Fracture Healing in NSAID TreatedRats

[0026] High resolution radiographs were made immediately post-fractureand then every week till the endpoint of the experiment (8 weeks) usinga Hewlett-Packard Faxitron. Shown are radiographs of the fractured rightfemurs from the same rats taken at 2 (top), 4 (middle), and 8 weeks(bottom) post-fracture (dorsal-ventral view). As indicated, panels A-Dshows radiographs from a no drug rat, an indomethacin treated rat, acelecoxib treated rat, and a rofecoxib treated rat, respectively. Notethat the fracture is still clearly evident in the 8-week post-fractureradiographs of the celecoxib and rofecoxib treated rats.

[0027]FIG. 2. COX-2-Selective NSAIDs Alter the Mechanical Properties ofFractured Femurs

[0028] Mechanical testing data was obtained or derived as described inthe Experimental Procedures. The data for each fractured femur wasnormalized as a percentage of the value obtained from that animal'scontralateral, unfractured femur, except for shear modulus panel D).Shown are the mean normalized values at each time point and for eachtreatment group or the mean shear modulus values in panel D. The errorbars represent standard errors of the mean. Pairwise t-test were madebetween the no drug and experimental treatments within a time point.Statistical significance differences (P<0.05) are noted with asterisks.

[0029]FIG. 3. COX-2-Selective NSAIDs Disrupt Endochondral OssificationDuring Fracture Healing

[0030] Shown are fracture calluses from no drug, indomethacin,celecoxib, and rofecoxib treated rats at 2, 3, and 4 weeks post-fractureas indicated. The specimens were embedded in polymethylmethacrylate(PMMA), sectioned, and stained with van Gieson's picrofuchsin andStevenel's blue so that bone is red, calcified cartilage is orange tored, cartilage is deep blue to purple, and fibrous tissue and muscle ispale blue. Each section is oriented with the cortical bone on thebottom, fracture callus on top, and fracture site in the middle. Notethe abnormal cartilage morphology in the calluses of the NSAID treatedrats (panels D, G, and J) and the lack of cartilage in the celecoxib(panels H and I) and rofecoxib treated (panels K and L) rats at 3 and 4weeks post-fracture.

[0031]FIG. 4. Abnormal Bone Resorption during Fracture Healing inCOX-2-Selective NSAID Treated Animals

[0032] Shown are fracture calluses from rofecoxib treated rats at 3(panels A and B) and 4 weeks (panel C) post-fracture. The orientation ofpanels A and B are the same with external callus on top and fracturesite to the immediate left of the panel. In panel C, the external callusis to the left and the fracture site is at the immediate bottom of thepanel. The specimens were embedded in PMMA, sectioned, and stained withvan Gieson's picrofuchsin and Stevenel's blue so that bone is red,calcified cartilage is orange to red, cartilage is deep blue to purple,and other cell types are shades of blue. Original photographicmagnification is indicated. CB: cortical bone; WB: woven bone; CC:calcified cartilage; Ca: cartilage; F: fracture site; ab: air bubble; M:area of magnification shown in panel B; and Oc: osteoclasts. The NSAIDtreated rats often developed areas of high bone resorption at thecortical bone, fracture site, external callus junction (M) as seen inpanel A. The air bubble (ab) seen in panel A is an artifact of the PMMAembedding. At higher magnification, osteoclasts (Oc) can be seen liningthe cortical bone surface of area M in the 3 week fracture callus asdenoted by the arrows. Shown in panel C is an identical area of a 4 weekpost-fracture callus as shown in panel B. The extent and area of boneresorption appears to be greater at 4 weeks post-fracture and also oftenencompassed all surfaces of the cortical bone at the fracture site.Similar bone resorption patterns were seen in celecoxib treated rats andto a lesser extent in the indomethacin treated rats.

[0033]FIG. 5. Experimental Complications Associated with NSAID Treatmentduring Fracture Healing

[0034] Complications that necessitated the pre-mature euthanization orresulted in the pre-mature death of a rat during the course of theseexperiments were compiled and used to determine the effects of NSAIDtreatment on anesthetic death, infection, and pin slippage. Experimentaltreatment group values were compared to the no drug values using a χ²analysis. Significant differences are noted with an asterisk (P<0.01).As can be seen, pin slippage was by far the most common complication andwas significantly different for each treatment group relative to the nodrug rats with P-values of less than 1E⁻⁴, 1E⁻⁷, and 1E⁻²⁴ for theindomethacin, celecoxib, and rofecoxib treated rats respectively. Therofecoxib treated rats were also found to have a statisticallysignificant higher infection rate as compared to the no drug rats(P<0.0001). Death from anesthesia was not different between groups.

[0035]FIG. 6. Cox2 but not Cox1 is Essential for Normal Bone FractureHealing

[0036] The right femora of Cox1^(−/−) and Cox2^(−/−) mice were fracturedand examined radiographically and histologically at 2 weekspost-fracture. Panels A and D. Radiographs of fractured femurs from aCox1^(−/−) and a Cox2^(−/−) mouse, respectively. Note the lack ofmineralized tissue (X-ray dense) in the fracture callus region of theCox2^(−/−) mouse. Panels B and C. Sagital section through the fracturedfemur of a Cox1^(−/−) stained with Masson's trichrome stain (cellnuclei=purple; muscle and cytoplasm=red; collagen and bone=blue). PanelsE and F. Sagital section through the fractured femur of a Cox2^(−/−)stained with Masson's trichrome stain. Note the presence of chondrocyteswithin the Cox2^(−/−) callus but the lack of endochondral ossificationrelative to the Cox1^(−/−) mouse fracture callus. Original photographicmagnification is indicated. The Cox2^(−/−) mouse fracture callusspecimens are shown at higher magnification because the callus wassmaller. B: bone; C: chondrocytes and cartilage; E: area of endochondralossification; M: area of intramembraneous bone formation; F: fracturesite.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention involves methods for enhancing woundhealing and treating conditions of the bone with the use ofcyclooxygenase (COX). COX is an enzyme that converts arachidonic acidinto prostaglandin H₂ (PGH₂). PGH₂ is then converted into specificprostaglandins, which affect bone metabolism and formation. There aretwo forms of cyclooxygenase, COX-1 and COX-2. COX-1 is constitutivelyexpressed, while COX-2 is inductively expressed by various stimuli.Examples of such stimuli are wounding and inflammation. The subsequentexpression of COX-2 in response to these stimuli results in theproduction of pro-inflammatory prostaglandins. The pain and inflammationassociated with a wound are commonly treated with non-steroidalanti-inflammatory drugs (NSAIDs). Most NSAIDs inhibit the cyclooxygenaseactivity of both COX-1 and COX-2. However, due to the inhibition ofCOX-1 activity, gastro-intestinal and kidney side effects result fromNSAIDs use. COX-2 selective NSAIDs have also been developed whichpreferentially inhibit COX-2 activity relative to COX-1 activity andthereby avoid the side effects associated with non-specific NSAIDs.

[0038] In addition to its inflammatory function, one aspect of theinvention relates to the necessity of COX-2 activity in wound healing.As described in more detail below, COX-2 is essential for normal woundhealing. In another aspect of the invention, treatment systems andmethods employ COX-2 to enhance wound healing.

[0039] In a preferred embodiment of the invention, gene therapytechniques are employed to increase cyclooxygenase activity at the siteof the wound to enhance wound healing. Gene therapy techniques allow anabsent or faulty gene to be replaced with a working gene. They alsoallow for the delivery and controlled expression of therapeutic geneproducts. One embodiment of the invention provides for a vectorcontaining a cyclooxygenase expression cassette. In a furtherembodiment, the vector containing the cyclooxygenase expression cassetteis delivered to the wound using gene therapy techniques. The genetherapy techniques may use adenoviral vectors, adeno-associated viralvectors, recombination-defective retrovirus vectors or DNA vectors todeliver the cyclooxygenase expression cassette to the wound.

[0040] The vectors of this invention may be used to increasecyclooxygenase levels at the site of the wound and thereby enhance wouldhealing. For example, these vectors may be used to enhance wound healingfollowing bone fractures or orthopaedic procedures. The vectors may alsobe of particular use to the elderly and other patient groups that havedelayed bone healing, including smokers, diabetics, and steroid users.In addition, the vectors may be used in the treatment of osteoporosisand osteogenesis imperfecta using gene therapy techniques for genereplacement and to enhance healing for wounds resulting from thesediseases.

[0041] One aspect of the invention involves a vector that contains acyclooxygenase expression cassette that encodes a cyclooxygenase geneproduct. The vector includes all necessary sequences for the expressionof the cyclooxygenase expression cassette and any sequences that may beincluded to control the expression of the cassette. These sequences mayinclude, but are not limited to, a promoter or initiation sequence, anenhancer sequence, termination sequence, RNA processing signals, and/ora polyadenylation signal sequence.

[0042] The term “vector” refers to a nucleic acid construct that encodesfor a particular gene product. The vectors of the present invention arepreferably adenoviral, adeno-associated viral, recombination-defectiveretrovirus, plasmid or DNA vectors.

[0043] The term “cyclooxygenase expression cassette” refers to nucleicacid which codes for the cyclooxygenase enzyme, preferably COX-1 orCOX-2. The expression cassette is positioned within the vector such thatit can be transcribed into RNA and translated into the cyclooxygenaseprotein product. Table 3 provides for the human DNA sequences for COX-1(SEQ. ID. NO. 1) and COX-2 (SEQ.ID. NO. 2).

[0044] The term “necessary sequences for the expression ofcyclooxygenase” refers to sequences necessary to ensure thetranscription and translation of the expression cassette. The term“promoter” refers to a DNA sequence that is bound by RNA polymerase andis required to initiate transcription of a gene. There are a number ofpromoters that are known in the art, including those that can enhance orcontrol expression of the gene or expression cassette. For example,cytomegalovirus promoter may be fused to the cyclooxygenase expressioncassette to obtain constitutive expression of cassette or a COX-2promoter may be linked to a cyclooxygenase cDNA to allow for expressionof cyclooxygenase in a more normal fashion. For example, a COX-2promoter may be linked to a COX-1 cDNA. This may allow a patient toremain on COX-2 selective NSAIDs but still have the ability to heal. Inanother aspect of the invention, the promoter may be induced in responseto inflammation. Preferably, the inducible promoter employs the use ofinflammatory gene promoters such as the interleukins, in particular, theIL-6 promoter, or the complement system gene promoters. Otherinflammatory gene promoters include promoters for TNF-α, or the NF-κBresponse element.

[0045] In another aspect of the invention, the vectors are delivereddirectly to the location of the wound by injection or direct applicationin order to enhance wound healing. The vectors of this invention mayalso be administered by electroporation or delivered as an aerosol. Thevectors may be administered or delivered in saline solutions,encapsulated in liposomes, in polymer solutions, in gels or lyophilized.In an alternative aspect, targeted transfection may be used to deliverthe vectors in vivo. The term “wound” refers to a bone fracture, thesite of a surgical orthopaedic procedure, or a skin lesion. In anotheraspect of the invention, the vectors may be delivered ex vivo, wherein apatient's cells are transfected ex vivo with the vectors of thisinvention and the transfected cells are then returned to the patient.

[0046] A further aspect of this invention provides for pharmaceuticalcompositions and methods of their use in wound healing. Thesepharmaceutical compositions include cyclooxgenase protein formulationsand/or pharmaceutically acceptable carriers. The cyclooxgenase proteinformulations comprise COX-1 protein, COX-2 protein or a combination ofthe two that are formulated such that the proteins remain stable, retaintheir function, including their enzymatic activity, and arephysiologically acceptable. The human amino acid sequences for COX-1(SEQ. ID. NO. 3) and COX-2 (SEQ. ID. NO. 4) proteins are provided inTable 3. There are a variety of pharmaceutically acceptable carriersthat are known in the art, including, but not limited to, saline,liposomes, gels and polymers. The formulation and/or carrier may also belyophilized for aerosol delivery. In a further embodiment of theinvention, these pharmaceutical compositions are administered topically,orally, intravenously, nasally or via inhalation, or locally for use inwound healing. Preferably, these composition are administered to enhancewound healing.

[0047] In another aspect of the invention, methods of enhancing woundhealing in the elderly are provided. COX-2 gene expression is reducedwith age as are wound healing and bone formation. In one embodiment,vectors containing cyclooxygenase expression cassettes are administeredto elderly to enhance would healing by increasing COX-2 expressionduring wound healing. A further aspect of this invention provides formethods for enhancing wound healing in patient groups that have delayedwound healing by delivering the vectors of this invention to thesepatients during wound healing.

[0048] In another embodiment of the invention, the vectors of thisinvention are used to enhance wound healing in patients suffering fromosteoporosis or OI by employing gene therapy techniques to delivercyclooxygenase expression cassettes to the wound to enhance healing.

[0049] In another aspect of the invention, a method for treatingpathological heterotopic ossification conditions is provided.Pathological heterotopic ossification conditions are diseasescharacterized by abnormal bone formation at locations of inflammation.An example of a pathological heterotopic ossification condition isfibrodysplasia ossificans progressiva (FOP). In one embodiment of theinvention, COX-2 selective NSAIDs are administered to locations ofinflammation that lack a wound. The COX-2 selective NSAIDs inhibit COX-2activity thereby preventing abnormal bone formation.

[0050] Our experimental approach was to assess fracture healing using astandard rat closed femur fracture model in which COX-2 function wasinhibited in vivo with the COX-2-selective NSAIDs, celecoxib androfecoxib. The results were striking in that femur fracture healing inrats treated with celecoxib or rofecoxib was dramatically impaired.These observations were confirmed by examining fracture healing in Cox1and Cox2 null mice. Histological observations indicated that the defectin fracture healing caused by the COX-2-selective NSAIDs or by lack ofCox2 occurred in the endochondral ossification pathway.

[0051] Using a standard rat closed femur fracture model, we havedemonstrated that COX-2-selective NSAID treatment can stop normalfracture healing and induce the formation of mal-unions and non-unions.Non-unions refer to fractures that show no visible sign of healing.Mal-unions refer to bones that do not heal properly and result inmisalignment of bones. These observations are in grim contrast to thoseobservations we, and others have obtained by treating rats withnon-selective NSAIDs such as indomethacin when it was observed thatfracture healing was delayed but not prevented (Rø et al. 1976; Rø etal. 1978). Our observations suggest that COX-2 has an essential functionduring normal fracture healing and that COX-2-selective NSAID inhibitionof prostaglandin synthesis stops normal fracture healing. This alsosuggests that the inflammatory phase is critical for normal fracturehealing.

[0052] Consistent with the effects of COX-2-selective NSAIDs on ratfemur fracture healing, mice homozygous for a targeted mutation in Cox2,but not Cox1, also showed inhibited fracture healing (FIG. 6). The DNAsequences (SEQ. ID. NO. 5 for COX-1 and SEQ. ID. NO. 6 for COX-2) andcorresponding amino acid sequences (SEQ. ID. NO. 7 for COX-1 and SEQ.ID. NO. 8 for COX-2) for mouse COX-1 and COX-2 gene and protein areprovided in Table 4. This excludes the possibility that any additionalinhibitory activity against other cellular processes or proteins bycelecoxib or rofecoxib (Jones et al. 1999; Hsu et al. 2000; Rossi et al.2000) is primarily responsible for negatively affecting fracturehealing.

[0053] The amount of rofecoxib used to treat the rats (3 mg/kg) in thisstudy was approximately 4 times the nominal, maximum human daily dose of50 mg (0.7 mg/kg) that is used to manage acute pain. In contrast, thecelecoxib dose used to treat rats in this study was in the recommendeddose range for humans. The rats received 280 mg/70 kg body weight ofcelecoxib once per day whereas the recommended human maximum daily doseof celecoxib is 200 mg twice a day. Additionally, the rats in this studyreceived daily doses of each drug till the endpoint of the experiment,which is unlike common clinical scenarios when COX-2-selective NSAIDsare used to acutely manage pain, inflammation, and swelling following afracture. In contrast, arthritis patients, in particular, do useCOX-2-selective NSAIDs on a daily basis for extended periods. Basedsimply upon animal body weight and drug dose, and without accounting forpharmacokinetic variables, the indomethacin dose used (1 mg/kg) would bepredicted to inhibit most COX-1 activity but to only partially inhibitCOX-2 activity; the celecoxib dose used should inhibit most if not allCOX-2 activity and possibly inhibit some COX-1 activity; and therofecoxib dose used should completely inhibit COX-2 activity but notaffect COX-1 (Warner et al. 1999). The estimated plasma half-lives forcelecoxib and rofecoxib in male rats following a single drug dose areapproximately 4 and 5 hours respectively (Halpin et al. 2000; Paulson etal. 2000). In humans, however, the estimated elimination half-life forcelecoxib is 11 hours (Davies et al. 2000) and the plasma half-life forrofecoxib is approximately 10-17 hours depending upon drug dose (Depréet al. 2000). Unfortunately, these data were unknown to us when thisstudy was initiated and consequently our rat drug dosing regime mayactually be an under representation of the COX-2 inhibition level over a24 hour cycle as that for humans receiving similar drug doses.Additionally, celecoxib and rofecoxib may have different inhibitoryconcentrations for rat versus human COX-2. Despite the pharmacokineticvariations between rats and humans receiving COX-2-selective NSAIDs, ourdata clearly indicate that these drugs have a dramatic negative effecton fracture healing in mammals, and thus caution in the use of theseCOX-2-selective NSAIDs in humans is warranted.

[0054] The fractured femurs of the celecoxib treated rats had increasedmechanical properties and healed as mal-unions, rather than non-unions,in approximately 50% of the rats tested (Tables 1 and 2). Had theexperimental endpoint been extended, some of the celecoxib treated ratsmay have gone on to heal their femur fractures. However, the delay inany such healing among the celecoxib treated rats would have beensignificantly longer than in no drug or indomethacin treated rats.

[0055] After mechanically testing the celecoxib and rofecoxib treatedfractured femurs, we observed that the fracture callus had a shell-likemorphology. The periphery of the fracture callus was bone that sometimespartly bridged the fracture gap and thus formed a mal-union in thecelecoxib treated rats. However, little or no bone was present betweenthe peripheral bone of the callus and the original femoral cortical boneends. Often this space appeared to be filled with fatty marrow. Alsostrikingly apparent was the lack of new bone or primary bone healing atthe cortical bone ends.

[0056] The indomethacin dose used in this study (1 mg/kg) was previouslyshown to delay fracture healing in rats and was used principally as apositive control (Altman et al. 1995). Increasing indomethacin doses tolevels that which would completely inhibit COX-2 (and COX-1 activity)causes a steep increase in rat mortality from gastrointestinal bleeding(Allen et al. 1980; Wallace et al. 1998). Consequently, it would bedifficult to directly compare the effects of COX-2-selective andtraditional NSAIDs on fracture healing based solely upon COX-2inhibition levels. An alternative approach would be to measureprostaglandin levels within and around the fracture callus duringhealing in control and drug treated rats and to then correlate thoseobservations with different healing parameters.

[0057] The abnormal osteoclastic response observed at the fracture sitein the NSAID treated rats is counter to experimental observations inwhich prostaglandins stimulate bone resorption (Klein and Raisz 1970)and COX-2 function promotes osteoclast formation (Okada et al. 2000).Lack of prostaglandins in the fracture callus could induce osteoclasticactivity through an undescribed mechanism, or perhaps through anindirect mechanism such as increased mechanical instability at thefracture site. An additional possibility is that the amount and/orrepertoire of prostaglandins produced at the fracture site in the NSAIDtreated rats is competent for inducing osteoclastic activity butinsufficient for osteogenesis to proceed normally. The secondpossibility is favored since the relative short half-life of celecoxiband rofecoxib in rats should produce daily periods in whichCOX-2-dependent prostaglandin synthesis could occur and because asimilar osteoclastic response was not observed in the Cox2^(−/−) mousefracture callus (FIG. 6). In addition, this large osteoclastic activitymay be the causative factor involved in fracture destabilization by pinslippage, which was the major morbidity complicating these experiments(FIG. 5).

[0058] Chondrocyte differentiation and persistence in the fracturecallus appears to be altered by COX-2-selective NSAID treatment and bylack of Cox2 (FIG. 6). Cartilage that is evident in the 2-week fracturecalluses of COX-2-selective NSAID treated rats either disappears or isdramatically reduced in the 3 and 4-week fracture calluses (FIG. 3). Incontrol rats, the fracture has bridged or is almost bridged by 4 weekspost-fracture as the cartilage present at early times had undergonenormal endochondral ossification and is no longer evident. In contrast,cartilage within the indomethacin treated fracture callus does persistat 4 weeks post-fracture indicating a reduced rate of endochondralossification. These observations suggest that the chondrocytes withinthe COX-2-selective NSAID treated rats deteriorated without forming acartilage matrix in which endochondral ossification could occur. Thisphenomenon then leads to the development of fracture non-unions andmal-unions. Similar histological observations were seen in theCox2^(−/−) mouse fracture where chondrocytes present in the callusfailed to form a mineralized matrix (FIG. 6). In support of thishypothesis, teratocarcinoma chondrocytes developed from Cox2^(−/−)embryonic stem cells were found to be deteriorating, hypotrophic, andundergoing apoptosis (Zhang et al. 2000). Additionally, the Cox2^(−/−)mouse fracture callus was much smaller than that from the Cox1^(−/−)mouse suggesting that mesenchymal cell proliferation and migration intothe Cox2^(−/−) mouse fracture site is reduced or that the mesenchymalcells proportionately differentiate into fewer chondrocytes. Thus, COX-2function is essential for normal progression of endochondralossification during fracture healing.

[0059] There are several steps during endochondral ossification at whichCOX-2 could exert an essential regulatory function. Endochondralossification is a complicated process that begins when chondrocytesmature to produce a cartilage matrix. Eventually the chondrocytes becomehypertrophic and undergo apoptosis as the cartilage matrix matures andbecomes calcified. Osteoclasts partially resorb the calcified cartilagewith concurrent angiogenesis at the site of endochondral ossification.Osteoblasts proliferate and differentiate on the calcified cartilage andbegin forming new bone. Remodeling of the new bone subsequently occursto increase the mechanical properties of the bone and restore its normalarchitecture. Prostaglandins produced by COX-2 could be used to enhanceosteoblast proliferation and differentiation (Kawaguchi et al. 1995).Prostaglandins may also be necessary to promote terminal differentiationof chondrocytes and formation of the cartilage matrix. Such an effect onchondrocytes could occur through a direct effect on differentiation orindirectly by preventing pre-mature apoptosis since inhibition of COX-2function by COX-2-selective NSAIDs has been shown to induce apoptosis incancer cell lines (Hsu et al. 2000). The potential effects ofprostaglandins on osteoblasts and chondrocytes during endochondralossification are not mutually exclusive. An additional possibility isthat COX-2 dependent signaling occurs between osteoblasts andchondrocytes to initiate and maintain endochondral ossification.Prostaglandins are also known to promote osteoclast activity, which maybe essential for the normal endochondral ossification process. However,our histological observations indicate an exuberant osteoclast responseat the fracture site in the NSAID treated rats thus we do not favor thispotential mechanism. Angiogenesis is also inhibited by COX-2-selectiveNSAIDs, at least in certain experimental models (Jones et al. 1999), andwe failed to detect neovascularization of the Cox2^(−/−) mouse fracturecallus. Thus an additional possibility is that lack of angiogenesisprecludes proper delivery of osteoclasts and osteoblasts to thecartilage matrix interface for continued endochondral ossification.

[0060] Our observations suggest that COX-2-selective NSAIDs may beeffective in reducing or preventing pathological heterotopicossification. One particular genetic disease for which COX-2-selectiveNSAIDs may be efficacious is fibrodysplasia ossificans progressiva(FOP)(Cohen et al. 1993). Children afflicted with FOP developdebilitating heterotopic bone through an endochondral ossificationpathway that appears to initiate at sites of inflammation (Kaplan et al.1993). Thus the COX-2-selective NSAIDs may be useful in reducinginflammation and stopping endochondral ossification at presumptiveheterotopic ossification sites in children with FOP.

[0061] To determine if COX-2 functions in fracture healing, rats weretreated with COX-2-selective non-steroidal anti-inflammatory drugs(NSAIDs) to stop COX-2-dependent prostaglandin production. Radiographic,histological, and mechanical testing demonstrated that fracture healingfailed in rats treated with COX-2 selective NSAIDs. Normal fracturehealing also failed in mice homozygous for a null mutation in the COX-2gene. These results demonstrate that COX-2 activity is necessary fornormal fracture healing and confirms that the effects of COX-2-selectiveNSAIDs on fracture healing is due to inhibition of COX-2 activity andnot from a drug side effect. Furthermore, histological observationssuggest that COX-2 is required for normal endochondral ossificationduring fracture healing. Since mice lacking Cox2 form normal skeletons,our observations indicate that fetal bone development and fracturehealing are different and that COX-2 function is specifically essentialfor fracture healing.

[0062] Experimental Procedures

[0063] Animals, Drug Dosage, and Administration

[0064] A total of 253 male Sprague-Dawley rats (584±62g) were fed astandard diet and kept caged separately in a constant temperature andhumidity environment. All rats were 6-9 months old at the beginning ofthe experiment. Drugs were administered daily by gavage beginning twodays prior to fracture. Animals were randomly selected for eachtreatment group. The rats were gavaged with aqueous suspensions ofindomethacin (1 mg/kg), celecoxib (4 mg/kg), or rofecoxib (3 mg/kg). Nodrug (control) rats were not initially gavaged but later rats weregavaged with water and no difference was noted between the no drug ratsthat had been gavaged and those that had not. No statisticallysignificant differences were found in animal weight changes during theexperiments.

[0065] Retired breeder female Cox1^(−/−) (B6;129P2-Ptgs1^(tm1)) andCox2^(−/−) (B6;129P2-Ptgs2^(tm1)) mice were obtained from Taconic Farms.Closed femur fracture production was done using a method similar to thatdescribed below.

[0066] Closed Femur Fracture Production

[0067] The rats were anesthetized by intraperitoneal injection ofketamine (40 mg/kg) and xylazine (5 mg/kg). Under aseptic conditions, amedial parapatellar incision (0.4-0.5 cm) was made in the right hindlimband the patella was dislocated laterally. The medullary canal wasentered through the intercondylar notch and reamed with an 18-gaugeneedle. A 1.1 mm stainless steel pin (Small Parts Inc., Miami Lakes,Fla.) was then inserted into the canal and secured in the proximal partof the greater trochanter by tamping. The distal portion of the pin wasthen cut flush with the femoral condyles and the patella dislocation wasreduced. The soft tissue and skin were closed with 4-0 vicryl sutures.After closing, the diaphysis of the pinned femur was fractured by meansof a three-point bending device as described by Bonnarens and Einhorn(Bonnarens and Einhorn 1984).

[0068] Radiography

[0069] Radiographs were made post-fracture to confirm the position andquality of each fracture and at sacrifice to determine the degree ofhealing. In addition several rats were selected randomly to produceserial radiographs (at least 2 rats per treatment group). Radiographswere made of these rats weekly under anesthesia until the experimentalendpoint (8 weeks). Radiographs of mice were also made under anesthesia.All radiographs were made using a 43805N Faxitron (Hewlett-Packard,McMinnville, Oreg.) and Kodak MinR-2000 mammography film.

[0070] Mechanical Testing

[0071] Animals within each treatment group were sacrificed at 4, 6, and8 weeks post-fracture by CO₂ asphyxiation. Animals with oblique,comminuted, or infected fractures were not used for mechanical testing.Both femora were removed and cleaned of all soft tissue leaving thefracture callus undisturbed and then immediately processed formechanical testing. The samples were wrapped in saline soaked gauze toprevent dehydration between steps. Measurements of the femora were takenusing digital calipers to determine femur length and external callusdimensions. The intramedullary pin was removed from the fractured femurand a 1 mm-diameter stainless steel pin (˜0.8 cm length) was inserted atthe proximal and distal end perpendicular to the long axis of the boneto prevent slipping in the potting material. The intact femur was alsopinned as described above. The femoral ends were potted in 1-inchhexnuts using a low melt temperature metal (Wood's metal, Alfa Aesar,Ward Mill, Mass.). Once potted, the gage length (L) of each femur wasmeasured. Torsional testing was conducted using a servohydraulic testingmachine (MTS, Eden, Praire, Minn.) with a 20 Nm reaction torque cell(Interface, Scottsdale, Ariz.). The testing was carried out to failureat a rate of 2°/sec and a data recording rate of 20 Hz. Both thefractured and intact femora were tested in internal rotation in properanatomic orientation. The peak torque and angle at failure werecalculated from the load-deformation curves. Internal fracture callusdimensions were measured after mechanical testing. From the callusdimensions, the polar moment of inertia (J) was calculated based upon ahollow ellipse model (Bell et al. 1941; Engesaeter et al. 1978). Theequations used to derive torsional rigidity, shear stress, shearmodulus, and J were as follows (Popov 1968): (1) Torsional Rigidity:(T_(max)·L)/φ where T_(max) is the peak torque value in Nmm, L is thegage length in mm, and σ is the angle at failure in radians. (2) ShearStress: (T_(max)·R_(max))/J where R_(max) is the largest radialdimension of the fracture callus in mm (a_(o)) and J is the polar momentof inertia. (3) Shear Modulus (G): (T_(max)·L)/J. (4) Polar Moment ofInertia (J): [π(ab³+a³b−(a−t)(b−t)³−(a−t)³(b−t)]/4 where a is[a_(i)+[(a_(o)−a_(i))/2]; b is [b_(i)+[(b_(o)−b_(i))/2]; t is theaverage bone thickness at the site of failure and is calculated as[(a_(o)−a_(i))+(b_(o)−b_(i))]/2 where a_(o) is the callus maximumoutside radius, a_(i) is the maximum interior radius, b_(o) is the leastoutside radius, and b_(i) is the least interior radius in mm. Onlytorsional testing data for which the fractured and control femur testedwithout incident were used.

[0072] Histology

[0073] Rats were sacrificed at 2, 3, 4, 6, and 8 weeks post-fracture byCO₂ asphyxiation. Both femora were resected and the stainless steel pinwas removed from the medullary canal. The harvested femora were fixed in10% buffered formalin and embedded in polymethylmethacrylate followingstandard histological techniques for calcified tissue. The samples weresectioned sagitally through the fracture callus using an Isomet diamondsaw (Buehler Ltd., Lake Bluff, Ill.), mounted on plexiglass slides, andpolished to a thickness of 100 μm. The slides were then stained with vanGieson's picrofuchsin and Stevenel's blue in order to identify new bonegrowth and cartilage formation (Maniatopoulos et al. 1986). Mice femorawere fixed, decalcified, paraffin embedded, sectioned, and stained withMasson's trichrome stain. The samples were viewed and photomicrographswere taken using an Olympus BH2-RFCA microscope or an Olympus SZ40microscope.

[0074] Treatment with COX-2-selective NSAIDs Leads to FractureNon-Unions and Mal-Unions

[0075] Femur fracture healing was followed by serial radiographicanalysis of rats treated with celecoxib, rofecoxib, indomethacin, orgavaged daily with water (no drugs group). Radiographs were madeimmediately following fracture production and then every week till theend point of the experiments (8 weeks post-fracture). Representativeresults are shown in FIG. 1.

[0076] We found that femur fracture healing proceeded normally in the nodrug rats as expected. At 1 week post-fracture, formation of the hardcallus could be detected radiographically but was more evident at 2weeks (FIG. 1A). By 4 weeks post-fracture, calcification of the softcallus was clearly evident indicating that endochondral ossification hadoccurred. Additionally, by 4 weeks post-fracture, the new bone formedduring fracture repair had almost bridged the fracture gap. Bridging ofthe fracture and remodeling of the fracture callus were evident at 6weeks post-fracture. Continued remodeling of the callus as well asremodeling of the original femoral cortical bone at the fracture site isclearly evident by 8 weeks post-fracture. These radiographicobservations are typical of normal fracture healing.

[0077] Indomethacin treatment appeared to delay but not prevent fracturehealing consistent with previous reports (Rø et al. 1976; Allen et al.1980; Altman et al. 1995). By 2 weeks post-fracture, an X-ray dense hardcallus is clearly evident in the indomethacin treated rats (FIG. 1B).However, bridging of the fracture gap did not appear to occur until 5-6weeks post fracture as compared to approximately 4-5 weeks post-fracturein the untreated rats (compare FIGS. 1A and B). Bridging and remodelingwere evident in the 8 week post-fracture radiographs of the indomethacintreated rats.

[0078] Celecoxib or rofecoxib treatment did not prevent formation of anX-ray dense hard callus as can be seen in the 2 and 4 week post-fractureradiographs (FIGS. 1C and D). However, the original fracture was stillplainly evident in the celecoxib (FIG. 1C) and rofecoxib (FIG. 1D)treated rats even after 8 weeks. No rofecoxib treated rat was observedto have a normally bridged callus by radiography. However, non-unions,mal-unions, and unions of the fractured femurs were observed byradiography in the celecoxib treated rats. The mal-unions were typifiedby the radiograph seen in FIG. 1C in which one cortex of the fracturecallus was bridged but in which the original cortical bone ends of thefractured femur had not joined and the fracture was still clearlyevident.

[0079] In addition to the serial radiographs made for certain rats, allanimals in this study were examined radiographically immediatelypost-fracture and when euthanized. A random, blinded sample of the8-week post-fracture radiographs were independently examined by 7observers and scored as a union (1 point), mal-union (0.5 points), ornon-union (0 points). Control rats had an average score of 0.71. Incontrast, the indomethacin, celecoxib and rofecoxib treated rats hadaverage scores of 0.54, 0.49, and 0.32, respectively. Despite the knowndifficulties associated with judging fracture healing from radiographs(Nicholls et al. 1979; Panjabi et al. 1989), a statistical comparisonbetween treatment groups showed that the no drug and rofecoxib groupswere significantly different (P<0.007 using a Fisher's PLSD test at a 5%significance level).

[0080] These observations clearly indicate that rofecoxib treatmentinhibits fracture healing in rats leading to non-unions. It would alsoappear that at least by radiographic examination, celecoxib treatmentnegatively affects fracture healing to an extent similar to, if notworse than, indomethacin treatment.

[0081] The Mechanical Properties of the Healing Femur Fracture Callusare Diminished by NSAID Treatment

[0082] In conjunction with the radiographic analysis, torsionalmechanical testing of fractured femurs was also performed. The fracturedfemur and contralateral control femur from rats at 4, 6, and 8 weekspost-fracture were tested to failure in torsion for each treatment group(no drugs, indomethacin, celecoxib, and rofecoxib). The data from thesetests is summarized in FIG. 2 and Table 1. Peak torque is the maximumtwisting force generated during torsional testing of the femur.Torsional rigidity is a measure of a structure's resistance to torque.Thus a bone with high torsional rigidity would fail after only a fewdegrees of rotation, but soft tissue would not reach its peak torqueuntil after a large angular deflection. Maximum shear stress is ameasure of the ultimate shearing force withstood by the femur prior tofailure and is a function of the applied torque and polar moment ofinertia, which is dictated by callus geometry. Shear modulus measuresthe elastic resistance to deformation by a shearing stress for a givenmaterial and is constant for a given material.

[0083] We found in the no drug rats that the normalized peak torque(101%) and torsional rigidity (88%) of the fractured femur was restoredby 8-weeks post-fracture as compared to the contralateral control femursfrom each animal (FIG. 2). However, the shear modulus (1.3 GPa) andnormalized shear stress (48%) of the fractured femurs at 8 weekspost-fracture were still less than the contralateral control femurs(FIG. 2, Table 1). This is the expected result because during fracturehealing, the ultimate mechanical integrity of the fractured bone, thatis peak torque, is maintained at a high level by increasing bonediameter via the fracture callus. Since the mechanical properties of theinitially soft tissue within the callus and later the newly formed boneare much weaker than the mechanical properties of mature cortical bone;shear stress and shear modulus were, as expected, less than thecontralateral control femurs. As the newly formed bone within thefracture callus matures by remodeling, the mechanical properties of thefractured bone increase. This is evident in our results as increases inshear stress and shear modulus with time (FIG. 2). The high normalizedtorsional rigidity found for the fractured femurs in the no drug rats at6 (89%) and 8 (88%) weeks post-fracture indicates that the fracture hadbeen bridged by new bone as would be expected.

[0084] We also observed that all of the 6 and 8 week post-fracture nodrug femurs and all the contralateral control femurs failed aspredicted, mid-diaphyseal spiral fractures during the torsionalmechanical testing.

[0085] Indomethacin treatment reduced the mechanical properties of thehealing femur fractures at earlier time points (FIG. 2). However by 8weeks post-fracture, the normalized peak torque, torsional rigidity, andshear stress values obtained from the indomethacin treated rats were notsignificantly different from the no drug rats. In contrast, at 6 weekspost-fracture, the normalized peak torque, torsional rigidity, and shearstress values (46, 34, and 15%, respectively) obtained from theindomethacin treated rats were less than the no drug rats at 6 weekspost-fracture (77, 89, and 36%, respectively). Pointedly, thesignificantly low torsional rigidity of the fractured femurs from theindomethacin treated rats at 6 weeks post-fracture indicates that thefracture had not been bridged by bone. Of the eight fractured femurstested at 8 weeks post-fracture, 6 failed as unions, 1 failed as amal-union, and 1 failed as a non-union (Table 2). These observationsindicate that the non-selective NSAID, indomethacin, delays, but doesnot prevent fracture healing, which is consistent with previous studiesand demonstrates the validity of our assay methods (Rø et al. 1976;Allen et al. 1980; Altman et al. 1995).

[0086] Rofecoxib treatment had a drastic effect on the mechanicalproperties of the fractured femurs. At 8 weeks post-fracture, for allvalues measured or derived, the mechanical properties of the fracturedfemurs from the rofecoxib treated rats were significantly less than theno drug rat fractured femurs (FIG. 2, Table 1). At 8 weekspost-fracture, the fractured femurs of the rofecoxib treated rats hadonly obtained 50%, 31%, and 18% of peak torque, torsional rigidity, ormaximum shear stress of the contralateral unfractured femurs,respectively. The low torsional rigidity and shear modulus (0.5 GPa)values obtained from the fractured femurs of the rofecoxib treated ratsare consistent with healing failure and the formation of non-unions. Inaddition, whereas all of the contralateral control femurs from therofecoxib treated rats failed as mid-diaphyseal spiral fractures, 4 ofthe 5 fractured femurs at 8 weeks post-fracture failed as non-unions andthe other failed as a malunion (Table 2). These observations demonstratethat, at the dose and treatment regime employed, the COX-2-selectiveNSAID rofecoxib stops fracture healing.

[0087] Unlike rofecoxib treatment, no significant differences were foundin the mechanical properties of the healing fractured femurs from thecelecoxib treated rats as compared to no drug rats. Despite the overallsimilarities in the mechanical values obtained between no drug rats andcelecoxib treated rats, 3 of 6 fractured femurs from the celecoxibtreated rats at 8 weeks post-fracture failed as non-unions during themechanical testing procedure and the other 3 failed as mal-unions (Table2). The relatively low normalized torsional rigidity (50%) and shearstress (22%) found for fractured femurs from the celecoxib treated ratsat 6 weeks post-fracture indicates that the fracture site had not beenbridged with bone (FIG. 2). Even though not statistically different fromthe no drug rat fractured femurs, the data obtained from the celecoxibtreated rat fractured femurs parallels closely the patterns obtainedfrom the femurs of the indomethacin treated rats. Together theseobservations suggest that, at the celecoxib dose and treatment regimeused, fracture healing is delayed and to a lesser extent than that foundfor the rofecoxib treatment regime, inhibited.

[0088] A χ² analysis was performed on visual inspection data obtainedfrom the 8 week post-fracture femurs following mechanical testing (Table2). The fractured femurs were considered to have failed as (a) unions ifa spiral fracture developed through the diaphysis of the femur, (b)non-unions if the femur failed completely along the original fracturesite, and (c) mal-unions if some new bone bridging of the fracture sitewas evident but that the femur still failed primarily along the originalfracture site. The data from the no drug, celecoxib, and rofecoxibtreatment groups were compared to that from the indomethacin treatmentgroup. Our analysis indicates that no statistical difference existsbetween the no drug and the indomethacin treatment groups but that thecelecoxib and rofecoxib treatment groups are significantly differentfrom the indomethacin treatment group. Again, these observationsstrongly indicate that inhibition of COX-2 dramatically inhibitsfracture healing.

[0089] No significant differences in the mechanical properties of thecontralateral femurs were found between treatment groups. This indicatesthat the experimental treatment regimes did not alter the intrinsicproperties of the rat bone by enhanced bone resorption or deposition atleast for the time frame examined.

[0090] COX-2-selective NSAID Treatment Alters Cartilage Formation DuringFracture Healing

[0091] The radiographic and torsional mechanic testing analyses of theCOX-2-selective NSAID treated rats demonstrated that these drugsdramatically inhibit fracture healing. However, hard callus formationappeared not to be impaired in the COX-2-selective NSAID treated rats.This suggests that COX-2-selective NSAIDs impair fracture healing in thesoft callus where endochondral ossification occurs. Since radiographycannot assess the early stages of endochondral ossification in the softcallus, we undertook a histological analysis of fracture healing in thedrug treated rats.

[0092] At 2 weeks post-fracture, gross abnormalities were present in thehistology of the healing femur fractures of the indomethacin, celecoxib,or rofecoxib treated rats as compared to an untreated control rat (FIG.3). In all four experimental groups, significant periostealintramembraneous ossification was evident at the fracture site asexpected from our radiographic data. Endochondral ossification alsoappeared to be proceeding normally in the no drug rat specimens. Incontrast, the histological specimens from the indomethacin andCOX-2-selective NSAID treated rats had abnormally formed cartilageelements within the callus. The positional extent of new bone formed inthe callus of the NSAID treated rats also appeared to be abnormal inthat it did not fully extend to the ends of the fractured bone. In theno drug rats, new bone in the callus extends to the very ends of thecortical bone fracture site. This is not so in the NSAID treated ratswhere this region of the callus is generally occupied by cartilage.

[0093] NSAID treatment grossly altered fracture callus morphology at 3and 4 weeks post-fracture (FIG. 3). Fracture healing proceeded normallyin the no drug rats with near bridging of the callus apparent by 4 weekspost-fracture in concurrence with our radiographic data (FIG. 1).However, healing was clearly delayed in the NSAID treated rats. Thefracture calluses of the indomethacin treated rats were not bridged at 4weeks but still appeared to be undergoing endochondral ossificationbased upon the presence of cartilage within the soft callus at 3 and 4weeks post-fracture. In contrast, little or no cartilage was evident inthe fracture calluses of the celecoxib or rofecoxib treated rats at 3and 4 weeks post-fracture indicating that endochondral ossification hadceased. Additionally massive resorption of the woven bone in the hardcallus of the NSAID treated rats appeared to leave a shell-like calluson the ends of the fractured bone.

[0094] Celecoxib and rofecoxib treatment often caused a massive boneresorption event at the distal ends of the fracture bones leaving whatappear to be indentations into the hard callus (FIG. 3 and 4). Themagnitude of this bone resorption phase is indicated by the large numberof osteoclasts that were found on the femur periosteal surface at thedistal ends of the fractured bone near the apparent indentation (FIG.4).

[0095] At 6 and 8 weeks post-fracture, the fractured femurs of the nodrug rats appeared to be healing normally with active remodeling of thecortical bone ends and fracture callus. Fractured femurs fromindomethacin treated rats also appeared to be healing at 6 and 8 weekspost-fracture with evident bridging and active remodeling. In contrast,no further healing was evident in the celecoxib or rofecoxib treated ratfractured femurs. The callus in the COX-2-selective NSAID treated ratswas smaller at 6 and 8 weeks post-fracture but the fracture gap wasstill clearly evident and often filled with fibrous tissue. Theseobservations are consistent with our radiographic and torsionalmechanical testing data demonstrating that celecoxib and rofecoxibinhibit fracture healing.

[0096] Complications Associated with Use of COX-2-Selective NSAIDs

[0097] As can be seen in FIG. 5, pin slippage was a severe complicationwith as many as 30% of the rofecoxib treated rats having to beeuthanized prior to the endpoint. Pin slippage is dislodgement of theintramedullary stainless steel rod used to stabilize the fracture andpermit the rat to weight-bear on the fractured femur. Once fracturestability was lost, the rat was euthanized since the rat could no longerweight-bear on the femur and since the callus would be re-injured andthus alter healing. The etiology of the pin slippage is unknown. Animalswith these complications were excluded. As such, final data may beskewed in favor of rats that had healed. Other complications includedanesthesia death during weekly radiographs and infections that excludedspecimens from further analysis. It was found using a χ² analysis tocompare each experimental group value to the no drug rat value that thepin slippage rate was significant for all NSAID treatment groups(P<0.0001) and that the infection rate for the rofecoxib treated ratswas also significant (P<0.0001).

[0098] Normal Fracture Healing Fails in Cox2 Null Mice

[0099] Our data clearly demonstrate that COX-2-selective NSAIDs aredetrimental to fracture healing. Unfortunately, these observations donot distinguish between a specific effect on COX-2 and a non-specificeffect of the NSAIDs on fracture healing. Therefore to specificallyaddress whether fracture healing requires Cox1 or Cox2 gene function,femur fracture healing was assessed in Cox1 and Cox2 knock-out mice(Langenbach et al. 1995; Morham et al. 1995). Using a modified method,closed femur fractures were produced in three female Cox1^(−/−) (Cox1knock-out) and three female Cox2^(−/−) (Cox2 knock-out) mice. Theanimals were examined radiographically immediately post-fracture andthen at 7, 10, 14, 21, 28, and 42 days post-fracture. Fracture healingappeared to proceed normally in the Cox1^(−/−) mice relative to ourprevious observations in outbred and inbred strains of mice (Manigrassoand O'Connor, unpublished). In contrast, only a slight periosteal hardcallus was detected in any of the Cox2^(−/−) mice, which is indicativeof healing failure. As can be seen in FIG. 6, the apparent difference infracture callus size was most obvious at 2 weeks post-fracture when theCox1^(−/−) mice had formed a large fracture callus but little or nocallus was evident in the Cox2^(−/−) mice.

[0100] One mouse of each genotype was euthanized at 2 weekspost-fracture and the fractured femur examined histologically (FIG. 6).New bone and differentiating chondrocytes were abundant within theCox1^(−/−) callus indicating that COX-1 activity is not essential forfracture healing. In contrast, there was a plainly evident lack of newbone formation in the Cox2^(−/−) fracture callus with only some apparentintramembraneous bone formation occurring at the edges of the callus(hard callus). Chondrocytes at different stages of differentiation wereobserved throughout the Cox2^(−/−) soft fracture callus. However, theCox2^(−/−) chondrocytes failed to form a mineralized matrix as evidentby the radiolucency and histological appearance of the soft callus. Theamount of endochondral ossification at the hard callus-soft callusboundary appeared greatly reduced in the Cox2^(−/−) specimen. Inaddition, neovascularization of the Cox2^(−/−) fracture callus was notobserved. These observations clearly indicate that normal fracturehealing and endochondral ossification are stopped or dramaticallyreduced in the Cox2^(−/−) mice and confirms our observations made in theCOX-2-selective NSAID treated rats.

[0101] Our data indicate that COX-2 function is essential for fracturehealing. In contrast, adult mice homozygous for targeted mutation ofCox2 appear to have normal skeletons. Together these observationsdemonstrate that fetal osteogenesis and fracture healing, though similarin many ways, are different and are probably initiated and maintainedthrough different molecular mechanisms. Cox2 is the first gene to beidentified that is specifically essential for fracture healing but notfetal osteogenesis. Targeted mutation of other mouse genes involved inprostaglandin synthesis and signaling should enable further analysis ofthe prostaglandin pathway(s) involved in fracture healing and skeletalbiology in general.

[0102] One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned as well as those inherent therein. Thecyclooxygenase vectors along with the methods, procedures and treatmentsdescribed herein are presently representative of preferred embodimentsand are exemplary and not intended as limitations on the scope of theinvention. Changes therein and other uses will occur to those skilled inthe art which are encompassed within the spirit of the invention ordefined by this scope with the claims.

[0103] It will be readily apparent to one skilled in the art thatvarying substitutions and modifications may be made to the inventiondisclosed herein within departing from the scope and spirit of theinvention.

[0104] All patents and publications referenced herein are incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.The following references are likewise incorporated by reference in orderto more fully describe the state of the art.

REFERENCES

[0105] Allen, H. L., A. Wase, and W. T. Bear. 1980. Indomethacin andaspirin: effect of nonsteroidal anti-inflammatory agents on the rate offracture repair in the rat. Acta. Orthop. Scand. 51: 595-600.

[0106] Altman, R. D., L. L. Latta, R. Keer, K. Renfree, F. J. Hornicek,and K. Banovac. 1995. Effect of nonsteroidal antiinflammatory drugs onfracture healing a laboratory study in rats. J. Orthop. Trauma 9:392-400.

[0107] Bell, G. H., D. P. Cuthbertson, and J. Orr. 1941. Strength andsize of bone in relation to calcium intake. Journal of Physiology 100:299-317.

[0108] Bolander, M. E. 1992. Regulation of fracture repair by growthfactors. Proc. Soc. Exp. Biol. Med. 200: 165-170.

[0109] Bonnarens, F. and T. A. Einhorn. 1984. Production of a standardclosed fracture in laboratory animal bone. J. Orthop. Res. 2: 97-101.

[0110] Brighton, C. T., J. L. Schaffer, D. B. Shapiro, J. J. S. Tang,and C. C. Clark. 1991. Proliferation and macromolecular synthesis by ratcalvarial bone cells grown in various oxygen tensions. J. Orthop. Res.9: 847-854.

[0111] Cohen, R. B., G. V. Hahn, J. A. Tabas, J. Peeper, C. L. Levitz,A. Sando, N. Sando, M. Zasloff, and F. S. Kaplan. 1993. The naturalhistory of heterotopic ossification in patients who have fibrodysplasiaossificans progressive. J. Bone and Joint Surg. 75-A: 215-219.

[0112] Davies, N. M., A. J. McLachlan, R. O. Day, and K. M. Williams.2000. Clinical pharmacokinetics and pharmacodynamics of celecoxib: aselective cyclo-oxygenase-2 inhibitor. Clinical Pharmacokinetics 38:225-242.

[0113] Dekel, S., G. Lenthall, and M. J. O. Francis. 1981. Release ofprostaglandins from bone and muscle after tibial fracture. J. Bone andJoint Surg. 63-B: 185-189.

[0114] Depré, M., E. Ehrich, A. van Hecken, I. De Lepeleire, A. Dallob,P. Wong, A. Porras, B. J. Gertz, and P. J. De Schepper. 2000.Pharmacokinetics, COX-2 specificity, and tolerability ofsupratherapeutic doses of rofecoxib in humans. European Journal ofPharmacology 56: 167-174.

[0115] Einhorn, T. A. 1998. The cell and molecular biology of fracturehealing. Clin Orthop: S7-21.

[0116] Engesaeter, L. B., A. Ekeland, and N. Langeland. 1978. Methodsfor testing the mechanical properties of the rat femur. Acta. Orthop.Scand. 49: 512-518.

[0117] Ferguson, C., E. Alpern, T. Miclau, and J. A. Helms. 1999. Doesadult fracture repair recapitulate embryonic skeletal formation? Mech.Dev. 87: 57-66.

[0118] Feyen, J. H., G. van der Wilt, P. Moonen, A. Di Bon, and P. J.Nijweide. 1984. Stimulation of arachidonic acid metabolism in primarycultures of osteoblast-like cells by hormones and drugs. Prostaglandins28: 769-781.

[0119] Halpin, R. A., L. A. Geer, K. E. Zhang, T. M. Marks, D. C. Dean,A. N. Jones, D. Melillo, G. Doss, and K. P. Vyas. 2000. The absorption,distribution, metabolism and excretion of rofecoxib, a potent andselective cyclooxygenase-2 inhibitor, in rats and dogs. Drug Metabolismand Disposition 28: 1244-1254.

[0120] Hsu, A.-L., T.-T. Ching, D.-S. Wang, X. Song, V. M. Rangnekar,and C.-S. Chen. 2000. The cyclooxygenase-2 inhibitor celecoxib inducesapoptosis by blocking Akt activation in human prostate cancer cellsindependently of Bc1-2. J. Biol. Chem. 275: 11397-11403.

[0121] Jones, M. K., H. Wang, B. M. Peskar, E. Levin, R. M. Itani, I. J.Sarfeh, and A. S. Tamawski. 1999. Inhibition of angiogenesis bynonsteroidal anti-inflammatory drugs: insight into the mechansims andimplications for cancer growth and ulcer healing. Nat. Med. 5:1418-1423.

[0122] Kaplan, F. S., J. A. Tabas, F. H. Gannon, G. Finkel, G. V. Hahn,and M. A. Zasloff. 1993. The histopathology of fibrodysplasia ossificansprogressiva: an endochondral process. J. Bone and Joint Surg. 75-A:220-230.

[0123] Kawaguchi, H., C. C. Pilbeam, J. R. Harrison, and L. G. Raisz.1995. The role of prostaglandins in the regulation of bone metabolism.Clin. Orth. Rel. Res. 313: 36-46.

[0124] Klein, D. C. and L. G. Raisz. 1970. Prostaglandins: stimulationof bone resorption in tissue culture. Endocrinol 86: 1436-1440.

[0125] Klein-Nulend, J., E. H. Burger, C. M. Semeins, L. G. Raisz, andC. C. Pilbeam. 1997. Pulsating fluid flow stimulates prostaglandinrelease and inducible prostaglandin G/H synthase mRNA expression inprimary mouse bone cells. J. Bone Miner. Res. 12: 45-51.

[0126] Langenbach, R., S. G. Morham, H. F. Tiano, C. D. Loftin, B. I.Ghanayem, P. C. Chulada, J. F. Mahler, C. A. Lee, E. H. Goulding, K. D.Kluckman, H. S. Kim, and O. Smithies. 1995. Prostaglandin synthase 1gene disruption in mice reduces arachidonic acid-induced inflammationand indomethacin-induced gastric ulceration. Cell 83: 483-492.

[0127] Maniatopoulos, C., A. Rodriguez, D. A. Deporter, and A. H.Melcher. 1986. An improved method for preparing histological sections ofmetallic implants. International Journal of Oral and MaxillofacialImplants 1: 31-37.

[0128] Masferrer, J. L., B. S. Zweifel, P. T. Manning, S. D. Hauser, K.M. Leahy, W. G. Smith, P. C. Isakson, and K. Seibert. 1994. Selectiveinhibition of inducible cyclooxygenase 2 in vivo is antiinflammatory andnonulcerogenic. Proc. Natl. Acad. Sci. USA 91: 3228-3232.

[0129] Moore, K. D., K. Goss, and J. O. Anglen. 1998. Indomethacinversus radiation therapy for prophylaxis against heterotopicossification in acetabular fractures. J. Bone and Joint Surg. 80-B:259-263.

[0130] Morham, S. G., R. Langenbach, C. D. Loftin, H. F. Tiano, N.Vouloumanos, J. C. Jennette, J. F. Mahler, K. D. Kluckman, A. Ledford,C. A. Lee, and O. Smithies. 1995. Prostaglandin synthase 2 genedisruption causes severe renal pathology in the mouse. Cell 83: 473-482.

[0131] Muscará, M. N., W. McKnight, S. Asfaha, and J. L. Wallace. 2000.Wound collagen deposition in rats: effects of an NO-NSAID and aselective COX-2 inhibitor. British Journal of Pharmacology 129: 681-686.

[0132] Needleman, P., J. Turk, B. A. Jakschik, A. R. Morrison, and J. B.Lefkowith. 1986. Arachidonic acid metabolism. Ann. Rev. Biochem. 55:69-102.

[0133] Nicholls, P. J., E. Berg, F. E. Bliven, Jr., and J. M. Kling.1979. X-ray diagnosis of healing fractures in rabbits. Clin. Orth. Rel.Res. 142: 234-236.

[0134] O'Banion, M. K., V. D. Winn, and D. A. Young. 1992. cDNA cloningand functional activity of a glucocorticoid-regulated inflammatorycyclooxygenase. Proc. Natl. Acad. Sci. USA 89: 4888-4892.

[0135] Okada, Y., J. A. Lorenzo, A. M. Freeman, M. Tomita, S. G. Morham,L. G. Raisz, and C. C. Pilbeam. 2000. Prostaglandin G/H synthase-2 isrequired for maximal formation of osteoclast-like cells in culture. J.Clin. Invest. 105: 823-832.

[0136] O'Sullivan, M. G., F. H. Chilton, E. M. Huggins, Jr., and C. E.McCall. 1992a. Lipopolysaccharide priming of alveolar macrophages forenhanced synthesis of prostanoids involves induction of a novelprostaglandin H synthase. J. Biol. Chem. 267: 14547-14550.

[0137] O'Sullivan, M. G., E. M. Huggins, Jr., E. A. Meade, D. L. DeWitt,and C. E. McCall. 1992b. Lipopolysaccharide induces prostaglandin Hsynthase-2 in alveolar macrophages. Biochem. Biophys. Res. Comm. 187:1123-1127.

[0138] Panjabi, M. M., R. W. Lindsey, S. D. Walter, and A. A. White,3rd. 1989. The clinician's ability to evaluate the strength of healingfractures from plain radiographs. J. Orthop. Trauma 3: 29-32.

[0139] Paulson, S. K., J. Y. Zhang, A. P. Breau, J. D. Hribar, N. W. K.Liu, S. M. Jessen, Y. M. Lawal, J. N. Cogburn, C. J. Gresk, C. S.Markos, T. J. Maziasz, G. L. Schoenhard, and E. G. Burton. 2000.Pharmacokinetics, tissue distribution, metabolism, and excretion ofcelecoxib in rats. Drug Metabolism and Disposition 28: 514-521.

[0140] Popov, E. P. 1968. Introduction to mechanics of solids.Prentice-Hall, Inc., Englewood Cliffs, N.J.

[0141] Pritchett, J. W. 1995. Ketorolac prophylaxis against heterotopicossification after hip replacement. Clin. Orth. Rel. Res. 314: 162-165.

[0142] Raskin, J. B. 1999. Gastrointestinal effects of nonsteroidalanti-inflammatory therapy. Am. J. Med. 106(5B): 3S-12S.

[0143] Riendeau, D., M. D. Percival, C. Brideau, S. Charleson, D. Dube,D. Ethier, J.-P. Falgueyret, R. W. Friesen, R. Gordon, G. Greig, J.Guay, J. Mancini, M. Ouellet, E. Wong, L. Xu, S. Boyce, D. Visco, Y.Girard, P. Prasit, R. Zamboni, I. W. Rodger, M. Gresser, A. W.Ford-Hutchinson, R. N. Young, and C.-C. Chan. 2001. Etoricoxib(MK-0663): preclinical profile and comparison with other agents thatselectively inhibit cyclooxygenase-2. Journal of Pharmacology andExperimental Therapeutics 296: 558-566.

[0144] Rø, J., N. Langeland, and J. Sander. 1978. Effect of indomethacinon collagen metabolism of rat fracture callus in vitro. Acta. Orthop.Scand. 49: 323-328.

[0145] Rø, J., E. Sudmann, and P. F. Marton. 1976. Effect ofindomethacin on fracture healing in rats. Acta. Orthop. Scand. 47:588-599.

[0146] Rossi, A., P. Kapahi, G. Natoli, T. Takahashi, Y. Chen, M. Karin,and M. G. Santoro. 2000. Anti-inflammatory cylopentenone prostaglandinsare direct inhibitors of IκB kinase. Nature 403: 103-108.

[0147] Shigeta, J.-I., S. Takahashi, and S. Okabe. 1998. Role ofcyclooxygenase-2 in the healing of gastric ulcers in rats. Journal ofPharmacology and Experimental Therapeutics 286: 1383-1390.

[0148] Topper, J. N., J. Cai, D. Falb, and M. A. Gimbrone, Jr. 1996.Identification of vascular endothelial genes differentially responsiveto fluid mechanical stimuli: cyclooxygenase-2, manganese superoxidedismutase, and endothelial cell nitric oxide synthase are selectivelyup-regulated by steady larninar shear stress. Proc. Natl. Acad. Sci. USA93: 10417-10422.

[0149] Vane, J. R., Y. S. Bakhle, and R. M. Botting. 1998.Cyclooxygenase 1 and 2. Annual Review of Pharmacology and Toxicology 38:97-120.

[0150] Vortkamp, A., S. Pathi, G. M. Peretti, E. M. Caruso, D. J.Zaleske, and C. J. Tabin. 1998. Recapitulation of signals regulatingembryonic bone formation during postnatal growth and fracture repair.Mech. Dev. 71: 65-76.

[0151] Wadleigh, D. J. and H. R. Herschman. 1999. Transcriptionalregulation of the cyclooxygenase-2 gene by diverse ligands in murineosteoblasts. Biochem. Biophys. Res. Comm. 264: 865-870.

[0152] Wallace, J. L., A. Bak, W. McKnight, S. Asfaha, K. A. Sharkey,and W. K. MacNaughton. 1998. Cyclooxygenase 1 contributes toinflammatory responses in rats and mice: implications forgastrointestinal toxicity. Gastroenterology 115: 101-109.

[0153] Warner, T. D., F. Giuliano, I. Vojnovic, A. Bukasa, J. A.Mitchell, and J. R. Vane. 1999. Nonsteroid drug selectivities forcyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated withhuman gastrointestinal toxicity: a full in vitro analysis. Proc. Natl.Acad. Sci. USA 96: 7563-7568.

[0154] Whelton, A. 1999. Nephrotoxicity of nonsteroidalanti-inflammatory drugs: physiological foundations and clinicalimplications. Am. J. Med. 106(5B): 13S-24S.

[0155] White, A. A., III, M. M. Panjabi, and W. O. Southwick. 1977. Thefour biomechanical stages of fracture repair. J. Bone and Joint Surg.59-A: 188-192.

[0156] Xie, W., J. G. Chipman, D. L. Robertson, R. L. Erikson, and D. L.Simmons. 1991. Expression of a mitogen-responsive gene encodingprostaglandin synthase is regulated by mRNA splicing. Proc. Natl. Acad.Sci. USA 88: 2692-2696.

[0157] Zhang, X., S. G. Morham, R. Langenbach, R. B. Baggs, and D. A.Young. 2000. Lack of cyclooxygenase-2 inhibits growth ofteratocarcinomas in mice. Exp. Cell Res. 254: 232-240. TABLE 1 TorsionalMechanical Testing Data Fractured Femur Data (not normalized tocontralateral control femur) All Contralateral No Drug IndomethacinCelecoxib Rofecoxib Control Femurs 4 wks 6 wks 8 wks 4 wks 6 wks 8 wks 4wks 6 wks 8 wks 4 wks 6 wks 8 wks Peak Torque (Tmax) in Nmm Mean 716 452430 643 417 357 469 302 567 620 250 383 338 CV 28 21 29 28 52 37 56 4434 49 53 33 36 Significance — — — — 0.69 0.36 0.16 0.03 0.15 0.88 0.010.54 0.01 Torsional Rigidity [(Tmax · L)/φ] Mean 83,050 27,200 66,06856,506 21,913 28,658 57,630 13,866 43,189 59,256 12,338 21,231 25,026 CV35 48 17 20 46 57 55 53 77 22 59 51 69 Significance — — — — 0.43 <0.010.93 0.06 0.11 0.69 0.04 <0.01 0.01 Shear Stress [(Tmax. · φ)/J] mean145 33 35 56 25 22 47 25 44 67 15 24 25 CV 38 42 34 27 85 47 66 73 73 4158 41 48 Significance — — — — 0.42 0.08 0.48 0.37 0.47 0.40 0.03 0.13<0.01 Shear Modulus (GPa) Mean 6.9 0.4 1.5 1.3 0.3 0.4 1.8 0.3 0.9 1.70.2 0.3 0.5 CV 54 58 71 38 78 73 99 77 116 27 49 56 91 Significance — —— — 0.33 0.08 0.46 0.26 0.40 0.16 0.04 0.07 0.01 Sample Size 81 6 5 7 87 8 8 8 6 6 7 5

[0158] TABLE 2 Frequency of Unions, Mal-Unions, and Non-Unions in theMechanically Tested Fractured Femurs at 8 weeks Post-Fracture UnionMal-Union Non-Union χ² (Stage IV)* (Stage III) (Stage I/Il) P-value** NoDrugs 7 0 0 0.338 Indomethacin 6 1 1 — Celecoxib 0 3 3 <0.001 Rofecoxib0 1 4 <0.001

[0159] TABLE 3 (SEQ. ID. NO.1) I. HUMAN COX-1 DNA SEQUENCEGCGCCATGAGCCGGAGTCTCTTGCTCCGGTTCTTGCTGTTCCTGCTCCTGCTCCCGCCGCTCCCCGTCCTGCTCGCGGACCCAGGGGCGCCCACGCCAGTGAATCCCTGTTGTTACTATCCATGCCAGCACCAGGGCATCTGTGTCCGCTTCGGCCTTGACCGCTACCAGTGTGACTGCACCCGCACGGGCTATTCCGGCCCCAACTGCACCATCCCTGGCCTGTGGACCTGGCTCCGGAATTCACTGCGGCCCAGCCCCTCTTTCACCCACTTCCTGCTCACTCACGGGCGCTGGTTCTGGGAGTTTGTCAATGCCACCTTCATCCGAGAGATGCTCATGCGCCTGGTACTCACAGTGCGCTCCAACCTTATCCCCAGTCCCCCCACCTACAACTCAGCACATGACTACATCAGCTGGGAGTCTTTCTCCAACGTGAGCTATTACACTCGTATTCTGCCCTCTGTGCCTAAAGATTGCCCCACACCCATGGGAACCAAAGGGAAGAAGCAGTTGCCAGATGCCCAGCTCCTGGCCCGCCGCTTCCTGCTCAGGAGGAAGTTCATACCTGACCCCCAAGGCACCAACCTCATGTTTGCCTTCTTTGCACAACACTTCACCCACCAGTTCTTCAAAACTTCTGGCAAGATGGGTCCTGGCTTCACCAAGGCCTTGGGCCATGGGGTAGACCTCGGCCACATTTATGGAGACAATCTGGAGCGTCAGTATCAACTGCGGCTCTTTAAGGATGGGAAACTCAAGTACCAGGTGCTGGATGGAGAAATGTACCCGCCCTCGGTAGAAGAGGCGCCTGTGTTGATGCACTACCCCCGAGGCATCCCGCCCCAGAGCCAGATGGCTGTGGGCCAGGAGGTGTTTGGGCTGCTTCCTGGGCTCATGCTGTATGCCACGCTCTGGCTACGTGAGCACAACCGTGTGTGTGACCTGCTGAAGGCTGAGCACCCCACCTGGGGCGATGAGCAGCTTTTCCAGACGACCCGCCTCATCCTCATAGGGGAGACCATCAAGATTGTCATCGAGGAGTACGTGCAGCAGCTGAGTGGCTATTTCCTGCAGCTGAAATTTGACCCAGAGCTGCTGTTCGGTGTCCAGTTCCAATACCGCAACCGCATTGCCATGGAGTTCAACCATCTCTACCACTGGCACCCCCTCATGCCTGACTCCTTCAAGGTGGGCTCCCAGGAGTACAGCTACGAGCAGTTCTTGTTCAACACCTCCATGTTGGTGGACTATGGGGTTGAGGCCCTGGTGGATGCCTTCTCTCGCCAGATTGCTGGCCGGATCGGTGGGGGCAGGAACATGGACCACCACATCCTGCATGTGGCTGTGGATGTCATCAGGGAGTCTCGGGAGATGCGGCTGCAGCCCTTCAATGAGTACCGCAAGAGGTTTGGCATGAAACCCTACACCTCCTTCCAGGAGCTCGTAGGAGAGAAGGAGATGGCAGCAGAGTTGGAGGAATTGTATGGAGACATTGATGCGTTGGAGTTCTACCCTGGACTGCTTCTTGAAAAGTGCCATCCAAACTCTATCTTTGGGGAGAGTATGATAGAGATTGGGGCTCCCTTTTCCCTCAAGGGTCTCCTAGGGAATCCCATCTGTTCTCCGGAGTACTGGAAGCCGAGCACATTTGGCGGCGAGGTGGGCTTTAACATTGTCAAGACGGCCACACTGAAGAAGCTGGTCTGCCTCAACACCAAGACCTGTCCCTACGTTTCCTTCCGTGTGCCGGATGCCAGTCAGGATGATGGGCCTGCTGTGGAGCGACCATCCACAGAGCTCTGAGGGGCAGGAAAGCAGCATTCTGGAGGGGAGAGCTTTGTGCTTGTCATTCCAGAGTGCTGAGGCCAGGGCTGATGGTCTTAAATGCTCATTTTCTGGTTTGGCATGGTGAGTGTTGGGGTTGACATTTAGAACTTTAAGTCTCACCCATTATCTGGAATATTGTGATTCTGTTTATTCTTCCAGAATGCTGAACTCCTTGTTAGCCCTTCAGATTGTTAGGAGTGGTTCTCATTTGGTCTGCCAGAATACTGGGTTCTTAGTTGACAACCTAGAATGTCAGATTTCTGGTTGATTTGTAACACAGTCATTCTAGGATGTGGAGCTACTGATGAAATCTGCTAGAAAGTTAGGGGGTTCTTATTTTGCATTCCAGAATCTTGACTTTCTGATTGGTGATTCAAAGTGTTGTGTTCCCTGGCTGATGATCCAGAACAGTGGCTCGTATCCCAAATCTGTCAGCATCTGGCTGTCTAGAATGTGGATTTGATTCATTTTCCTGTTCAGTGAGATATCATAGAGACGGAGATCCTAAGGTCCAACAAGAATGCATTCCCTGAATCTGTGCCTGCACTGAGAGGGCAAGGAAGTGGGGTGTTCTTCTTGGGACCCCCACTAAGACCCTGGTCTGAGGATGTAGAGAGAACAGGTGGGCTGTATTCACGCCATTGGTTGGAAGCTACCAGAGCTCTATCCCCATCCAGGTCTTGACTCATGGCAGCTGTTTCTCATGAAGCTAATAAAATTC GCCC (SEQ. ID. NO.2)II. HUMAN COX-2 DNA SEQUENCECAATTGTCATACGACTTGCAGTGAGCGTCAGGAGCACGTCCAGGAACTCCTCAGCAGCGCCTCCTTCAGCTCCACAGCCAGACGCCCTCAGACAGCAAAGCCTACCCCCGCGCCGCGCCCTGCCCGCCGCTCGGATGCTCGCCCGCGCCCTGCTGCTGTGCGCGGTCCTGGCGCTCAGCCATACAGCAAATCCTTGCTGTTCCCACCCATGTCAAAACCGAGGTGTATGTATGAGTGTGGGATTTGACCAGTATAAGTGCGATTGTACCCGGACAGGATTCTATGGAGAAAACTGCTCAACACCGGAATTTTTGACAAGAATAAAATTATTTCTGAAACCCACTCCAAACACAGTGCACTACATACTTACCCACTTCAAGGGATTTTGGAACGTTGTGAATAACATTCCCTTCCTTCGAAATGCAATTATGAGTTATGTCTTGACATCCAGATCACATTTGATTGACAGTCCACCAACTTACAATGCTGACTATGGCTACAAAAGCTGGGAAGCCTTCTCTAACCTCTCCTATTATACTAGAGCCCTTCCTCCTGTGCCTGATGATTGCCCGACTCCCTTGGGTGTCAAAGGTAAAAAGCAGCTTCCTGATTCAAATGAGATTGTGGAAAAATTGCTTCTAAGAAGAAAGTTCATCCCTGATCCCCAGGGCTCAAACATGATGTTTGCATTCTTTGCCCAGCACTTCACGCATCAGTTTTTCAAGACAGATCATAAGCGAGGGCCAGCTTTCACCAACGGGCTGGGCCATGGGGTGGACTTAAATCATATTTACGGTGAAACTCTGGCTAGACAGCGTAAACTGCGCCTTTTCAAGGATGGAAAAATGAAATATCAGATAATTGATGGAGAGATGTATCCTCCCACAGTCAAAGATACTCAGGCAGAGATGATCTACCCTCCTCAAGTCCCTGAGCATCTACGGTTTGCTGTGGGGCAGGAGGTCTTTGGTCTGGTGCCTGGTCTGATGATGTATGCCACAATCTGGCTGCGGGAACACAACAGAGTATGCGATGTGCTTAAACAGGAGCATCCTGAATGGGGTGATGAGCAGTTGTTCCAGACAAGCAGGCTAATACTGATAGGAGAGACTATTAAGATTGTGATTGAAGATTATGTGCAACACTTGAGTGGCTATCACTTCAAACTGAAATTTGACCCAGAACTACTTTTCAACAAACAATTCCAGTACCAAAATCGTATTGCTGCTGAATTTAACACCCTCTATCACTGGCATCCCCTTCTGCCTGACACCTTTCAAATTCATGACCAGAAATACAACTATCAACAGTTTATCTACAACAACTCTATATTGCTGGAACATGGAATTACCCAGTTTGTTGAATCATTCACCAGGCAAATTGCTGGCAGGGTTGCTGGTGGTAGGAATGTTCCACCCGCAGTACAGAAAGTATCACAGGCTTCCATTGACCAGAGCAGGCAGATGAAATACCAGTCTTTTAATGAGTACCGCAAACGCTTTATGCTGAAGCCCTATGAATCATTTGAAGAACTTACAGGAGAAAAGGAAATGTCTGCAGAGTTGGAAGCACTCTATGGTGACATCGATGCTGTGGAGCTGTATCCTGCCCTTCTGGTAGAAAAGCCTCGGCCAGATGCCATCTTTGGTGAAACCATGGTAGAAGTTGGAGCACCATTCTCCTTGAAAGGACTTATGGGTAATGTTATATGTTCTCCTGCCTACTGGAAGCCAAGCACTTTTGGTGGAGAAGTGGGTTTTCAAATCATCAACACTGCCTCAATTCAGTCTCTCATCTGCAATAACGTGAAGGGCTGTCCCTTTACTTCATTCAGTGTTCCAGATCCAGAGCTCATTAAAACAGTCACCATCAATGCAAGTTCTTCCCGCTCCGGACTAGATGATATCAATCCCACAGTACTACTAAAAGAACGTTCGACTGAACTGTAGAAGTCTAATGATCATATTTATTTATTTATATGAACCATGTCTATTAATTTAATTATTTAATAATATTTATATTAAACTCCTTATGTTACTTAACATCTTCTGTAACAGAAGTCAGTACTCCTGTTGCGGAGAAAGGAGTCATACTTGTGAAGACTTTTATGTCACTACTCTAAAGATTTTGCTGTTGCTGTTAAGTTTGGAAAACAGTTTTTATTCTGTTTTATAAACCAGAGAGAAATGAGTTTTGACGTCTTTTTACTTGAATTACTATCACAAGATGGCAAAATGCTGAAAGTTTTTACACTGTCGATGTTTCCAATGCATCTTCCATGATGCATTAGAAGTAACTAATGTTTGAAATTTTAAAGTACTTTTGGTTATTTTTCTGTCATCAAACAAAAACAGGTATCAGTGCATTATTAAATGAATATTTAAATTAGACATTACCAGTAATTTCATGTCTACTTTTTAAAATCAGCAATGAAACAATAATTTGAAATTTCTAAATTCATAGGGTAGAATCACCTGTAAAAGCTTGTTTGATTTCTTAAAGTTATTAAACTTGTACATATACCAAAAAGAAGCTGTCTTGGATTTAAATCTGTAAAATCAGATGAAATTTTACTACAATTGCTTGTTAAAATATTTTATAAGTGATGTTCCTTTTTCACCAAGAGTATAAACCTTTTTAGTGTGACTGTTAAAACTTCCTTTTAAATCAAAATGCCAAATTTATTAAGGTGGTGGAGCCACTGCAGTGTTATCTCAAAATAAGAATATTTTGTTGAGATATTCCAGAATTTGTTTATATGGCTGGTAACATGTAAAATCTATATCAGCAAAAGGGTCTACCTTTAAAATAAGCAATAACAAAGAAGAAAACCAAATTATTGTTCAAATTTAGGTTTAAACTTTTGAAGCAAACTTTTTTTTATCCTTGTGCACTGCAGGCCTGGTACTCAGATTTTGCTATGAGGTTAATGAAGTACCAAGCTGTGCTTGAATAACGATATGTTTTCTCAGATTTTCTGTTGTACAGTTTAATTTAGCAGTCCATATCACATTGCAAAAGTAGCAATGACCTCATAAAATACCTCTTCAAAATGCTTAAATTCATTTCACACATTAATTTTATCTCAGTCTTGAAGCCAATTCAGTAGGTGCATTGGAATCAAGCCTGGCTACCTGCATGCTGTTCCTTTTCTTTTCTTCTTTTAGCCATTTTGCTAAGAGACACAGTCTTCTCATCACTTCGTTTCTCCTATTTTGTTTTACTAGTTTTAAGATCAGAGTTCACTTTCTTTGGACTCTGCCTATATTTTCTTACCTGAACTTTTGCAAGTTTTCAGGTAAACCTCAGCTCAGGACTGCTATTTAGCTCCTCTTAAGAAGATTAAAAGAGAAAAAAAAAGGCCCTTTTAAAAATAGTATACACTTATTTTAAGTGAAAAGCAGAGAATTTTATTTATAGCTAATTTTAGCTATCTGTAACCAAGATGGATGCAAAGAGGCTAGTGCCTCAGAGAGAACGCCCTTTCTTATTTAAAAACAAAACCAAATGATATCTAAGTAGTTCTCAGCAATAATAATAATGACGATAATACTTCTTTTCCACATCTCATTGTCACTGACATTTAATGGTACTGTATATTACTTAATTTATTGAAGATTATTATTTATGTCTTATTAGGACACTATGGTTATAAACTGTGTTTAAGCCTACAATCATTGATTTTTTTTTGTTATGTCACAATCAGTATATTTTCTTTGGGGTTACCTCTCTGAATATTATGTAAACAATCCAAAGAAATGATTGTATTAAGATTTGTGAATAAATTTTTAGAAATCTGATTGGCATATTGAGATATTTAAGGTTGAATGTTTGTCCTTAGGATAGGCCTATGTGCTAGCCCACAAAGAATATTGTCTCATTAGCCTGAATGTGCCATAAGACTGACCTTTTAAAATGTTTTGAGGGATCTGTGGATGCTTCGTTAATTTGTTCAGCCACAATTTATTGAGAAAATATTCTGTGTCAAGCACTGTGGGTTTTAATATTTTTAAATCAAACGCTGATTACAGATAATAGTATTTATATAAATAATTGAAAAAATTTTCTTTTGGGAAGAGGGAGAAAATGAAATAAATATCATTAAAGATAACTCAGGAGAATCTTCTTTACAATTTTACGTTTAGAATGTTTAAGGTTAAGAAAGAAATAGTCAATATGCTTGTATAAAACACTGTTCACTGTTTTTTTTAAAAAAAAAACTTGATTTGTTATTAACATTGATCTGCTGACAAAACCTGGGAATTTGGGTTGTGTATGCGAATGTTTCAGTGCCTCAGACAAATGTGTATTTAACTTATGTAAAAGATAAGTCTGGAAATAAATGTCTGT TTATTTTTGTACTATTTA(SEQ. ID. NO.3) III. HUMAN COX-1 AMINO ACID SEQUENCEMSRSLLLRFLLFLLLLPPLPVLLADPGAPTPVNPCCYYPCQHQGICVRFGLDRYQCDCTRTGYSGPNCTIPGLWTWLRNSLRPSPSFTHFLLTFGRWFWEFVNATFIREMLMRLVLTVRSNLIPSPPTYNSAHDYISWESFSNVSYYTRILPSVPKDCPTPMGTKGKKQLPDAQLLARRFLLRRKFIPDPQGTNLMFAFFAQHFTHQFFKTSGKMGPGFTKALGHGVDLGHIYGDNLERQYQLRLFKDGKLKYQVLDGEMYPPSVEEAPVLMHYPRGIPPQSQMAVGQEVFGLLPGLMLYATLWLREHNRVCDLLKAEHPTWGDEQLFQTTRLILIGETIKIVIEEYVQQLSGYFLQLKFDPELLFGVQFQYRNRIAMEFNHLYHWHPLMPDSFKVGSQEYSYEQFLFNTSMLVDYGVEALVDAFSRQIAGRIGGGRNMDHHILHVAVDVIRESREMRLQPFNEYRKRFGMKPYTSFQELVGEKEMAAELEELYGDIDALEFYPGLLLEKCHPNSIFGESMIEIGAPFSLKGLLGNPICSPEYWKPSTFGGEVGFNIVKTATLKKLVCLNTKTCPYVSFRVPDASQDDGPAVERPSTEL (SEQ. ID. NO.4) IV.HUMAN COX-2 AMINO ACID SEQUENCEMLARALLLCAVLALSHTANPCCSHPCQNRGVCMSVGFDQYKCDCTRTGFYGENCSTPEFLTRIKLFLKPTPNTVHYILTHFKGFWNVVNNIPFLRNAIMSYVLTSRSHLIDSPPTYNADYGYKSWEAFSNLSYYTRALPPVPDDCPTPLGVKGKKQLPDSNEIVEKLLLRRKFIPDPQGSNMMFAFFAQHFTHQFFKTDHKRGPAFTNGLGHGVDLNHIYGETLARQRKLRLFKDGKMKYQIIDGEMYPPTVKDTQAEMIYPPQVPEHLRFAVGQEVFGLVPGLMMYATIWLREHNRVCDVLKQEHPEWGDEQLFQTSRLILIGETIKIVIEDYVQHLSGYHFKLKFDPELLFNKQFQYQNRIAAEFNTLYHWHPLLPDTFQIHDQKYNYQQFIYNNSILLEHGITQFVESFTRQIAGRVAGGRNVPPAVQKVSQASIDQSRQMKYQSFNEYRKRFMLKPYESFEELTGEKEMSAELEALYGDIDAVELYPALLVEKPRPDAIFGETMVEVGAPFSLKGLMGNVICSPAYWKPSTFGGEVGFQIINTASIQSLICNNVKGCPFTSFSVPDPELIKTVTINASSSRSGLDDINPTVLLKER STEL

[0160] TABLE 4 (SEQ. ID. NO.5) I. MOUSE COX-1 DNA SEQUENCEGCCGTTGGCATTGCACATCCATCCACTCCCAGAGTCATGAGTCGAAGGAGTCTCTCGCTCTGGTTTCCCCTGCTGCTGCTCCTGCTGCTGCCGCCGACACCCTCGGTCCTGCTCGCAGATCCTGGGGTGCCCTCACCAGTCAATCCCTGTTGTTACTATCCGTGCCAGAACCAGGGTGTCTGTGTCCGCTTTGGCCTCGACAACTACCAGTGTGATTGTACTCGCACGGGCTACTCAGGCCCCAACTGTACCATCCCTGAGATCTGGACCTGGCTTCGGAATTCTCTGCGGCCCAGCCCCTCGTTCACCCATTTCCTGCTGACACATGGATACTGGCTCTGGGAATTTGTGAATGCCACCTTCATCCGAGAAGTACTCATGCGCCTGGTACTCACAGTGCGGTCCAACCTTATCCCCAGCCCTCCGACCTACAACTCAGCGCATGACTACATCAGCTGGGAGTCCTTCTCCAATGTGAGCTACTATACTCGCATTCTGCCCTCTGTACCCAAAGACTGCCCCACACCCATGGGGACCAAAGGGAAGAAACAGTTACCAGATGTTCAGCTTCTGGCCCAACAGCTGCTGCTGAGAAGGGAGTTCATTCCTGCCCCCCAGGGCACCAACATCCTGTTTGCCTTCTTTGCACAACACTTCACCCACCAGTTCTTCAAGACCTCTGGAAAGATGGGTCCTGGCTTTACCAAGGCCTTAGGCCACGGGGTAGACCTTGGCCACATTTATGGAGATAATCTGGAACGACAGTATCACCTGCGGCTCTTCAAGGATGGGAAACTTAAGTACCAGGTGCTGGACGGAGAGGTGTACCCACCTTCCGTGGAACAGGCGTCCGTGTTGATGCGCTACCCACCAGGTGTCCCGCCTGAAAGGCAGATGGCTGTGGGCCAGGAGGTGTTTGGGTTGCTTCCGGGGCTGATGCTCTTCTCCACGATCTGGCTTCGTGAACATAACCGCGTGTGCGACCTGCTGAAGGAGGAGCATCCCACGTGGGATGATGAGCAGCTCTTCCAGACCACTCGCCTCATCCTTATAGGAGAAACCATCAAAATTGTCATTGAGGAGTATGTGCAGCACTTGAGTGGCTATTTCCTGCAGCTCAAGTTTGACCCGGAGCTGCTGTTCCGAGCCCAGTTCCAATATCGAAACCGCATCGCCATGGAATTTAACCATCTCTATCACTGGCATCCACTCATGCCCAACTCCTTCCAAGTGGGCTCACAAGAGTACAGCTACGAGCAGTTTTTATTTAACACTTCTATGCTGGTGGACTATGGGGTTGAGGCACTGGTGGATGCCTTCTCTCGCCAGAGGGCTGGCCGGATTGGTGGAGGTAGGAACTTTGACTATCATGTTCTGCATGTGGCTGTGGATGTCATCAAGGAGTCCCGAGAGATGCGCCTACAGCCCTTCAATGAATACCGAAAGAGGTTTGGCTTGAAGCCTTACACCTCTTTCCAGGAGCTCACAGGAGAGAAGGAGATGGCTGCTGAGTTGGAGGAGCTGTACGGTGACATCGATGCTTTAGAGTTCTACCCGGGGTTGCTGCTGGAGAAGTGCCAGCCCAACTCCATCTTTGGAGAAAGTATGATAGAGATGGGGGCTCCCTTTTCCCTCAAGGGCCTCCTAGGGAATCCCATCTGTTCCCCAGAGTACTGGAAACCCAGCACGTTCGGTGGTGACGTGGGCTTCAACCTTGTCAACACAGCCTCACTGAAGAAACTGGTCTGCCTCAACACCAAGACCTGCCCCTATGTTTCCTTCCGTGTGCCAGATTACCCTGGAGATGACGGGTCTGTCTTAGTGAGACGCTCCACTGAGCTCTGAGGGAGCTGGAAAGCAGCCTCTGGAGGGAGGAGTTTTGTTCCTGATGAAGACAAGTCCTTGATGTGGGTTTTCGTGGCTTGGCATTGTGAGAGCTGATGCTCACATTTGAAACTTTGGGTCTTACCCTTGCCTAGAAAATTGTGATTTTGCCACTTTCGGATGTTGAATTCTTTGTTAACTAAGAAAGTTAGAAGTGGTTTTGTCTGCCTCCTCAGAACTTGGCTCTTTGTTGGCAACTCAGAAAGTCAGATTTCTGGTTGATTTGGAATATAGGCTTAAAACTTTATATTATAGGGTAGGGTGTGGTTGCACACACCTTAATCCCAGCACTTGGAAGGCAGAGGCAGTTGGATCTCTGGGAGTTTGAGGCCAGTTTGGCCTATATAGTGAGTTCTAGGCCAGCCATGGATGCATAGTGAGACTCTTTCTCAAAACAAACAAACAAACAAACAAACAAACAACTTTTAGAATGTAGAATTCCGTAAAAAAAAAAAATCCCTTGAAAGTTAATGGGGTCCTCAATTTTCTTCCTAGAATTTGGAGGCCTCTTCAGAATGTTGACTATCTGACAGGTGACTCAGAAGGTCCTGTTCCTGGTCAATGATCTATAACATGGGCCAAAACATTCCCAACTTGAATGTCTAGAATGTGGAATTGGTTCATTTTCCTGTTCAGTGAAATGGACACAGAACAAAAGAACCCAGTGTCCAGCAAGAATTGCCTTGCCCAAACCTACGTCTACGCCAAAGGTCAAGGCAGTAAGGTGTTCTTGGGAGCCACACTTAGACTCTTTCCAAAGATGTGGAGGGAACAGATGGACTCATCTATGATCTTGGTTGGAAACCACCACAGTTCTATCCCCATCCAGATCTTTGCTTGTGGCAGCTGTTTCTCATGAAGCTAAT AAAATTC (SEQ. ID.NO.6) II. MOUSE COX-2 DNA SEQUENCEAGTTGTCAAACTGCGAGCTAAGAGCTTCAGGAGTCAGTCAGGACTCTGCTCACGAAGAATCTCAGCACTGCATCCTGCCAGCTCCACCGCCACCACCACTGCCACCTCCGCTGCCACCTCTGCGATGCTCTTCCGAGCTGTGCTGCTCTGCGCTGCCCTGGGGCTCAGCCAGGCAGCAAATCCTTGCTGTTCCAATCCATGTCAAAACCGTGGGGAATGTATGAGCACAGGATTTGACCAGTATAAGTGTGACTGTACCCGGACTGGATTCTATGGTGAAAACTGTACTACACCTGAATTTCTGACAAGAATCAAATTACTGCTGAAGCCCACCCCAAACACAGTGCACTACATCCTGACCCACTTCAAGGGAGTCTGGAACATTGTGAACAACATCCCCTTCCTGCGAAGTTTAACTATGAAATATGTGCTGACATCCAGATCATATTTGATTGACAGTCCACCTACTTACAATGTGCACTATGGTTACAAAAGCTGGGAAGCCTTCTCCAACCTCTCCTACTACACCAGGGCCCTTCCTCCAGTAGCAGATGACTGCCCAACTCCCATGGGTGTGAAGGGAAATAAGGAGCTTCCTGATTCAAAAGAAGTGCTGGAAAAGGTTCTTCTACGGAGAGAGTTCATCCCTGACCCCCAAGGCTCAAATATGATGTTTGCATTCTTTGCCCAGCACTTCACCCATCAGTTTTTCAAGACAGATCATAAGCGAGGACCTGGGTTCACCCGAGGACTGGGCCATGGAGTGGACTTAAATCACATTTATGGTGAAACTCTGGACAGACAACATAAACTGCGCCTTTTCAAGGATGGAAAATTGAAATATCAGGTCATTGGTGGAGAGGTGTATCCCCCCACAGTCAAAGACACTCAGGTAGAGATGATCTACCCTCCTCACATCCCTGAGAACCTGCAGTTTGCTGTGGGGCAGGAAGTCTTTGGTCTGGTGCCTGGTCTGATGATGTATGCCACCATCTGGCTTCGGGAGCACAACAGAGTGTGCGACATACTCAAGCAGGAGCATCCTGAGTGGGGTGATGAGCAACTATTCCAAACCAGCAGACTCATACTCATAGGAGAGACTATCAAGATAGTGATCGAAGACTACGTGCAACACCTGAGCGGTTACCACTTCAAACTCAAGTTTGACCCAGAGCTCCTTTTCAACCAGCAGTTCCAGTATCAGAACCGCATTGCCTCTGAATTCAACACACTCTATCACTGGCACCCCCTGCTGCCCGACACCTTCAACATTGAAGACCAGGAGTACAGCTTCAAACAGTTTCTCTACAACAACTCCATCCTCCTGGAACATGGACTCACTCAGTTTGTTGAGTCATTCACCAGACAGATTGCTGGCCGGGTTGCTGGGGGAAGAAATGTGCCAATTGCTGTACAAGCAGTGGCAAAGGCCTCCATTGACCAGAGCAGAGAGATGAAATACCAGTCTCTCAATGAGTACCGCAAACGCTTCTCCCTGAAGCCGTACACATCATTTGAAGAACTTACAGGAGAGAAGGAAATGGCTGCAGAATTGAAAGCCCTCTACAGTGACATCGATGTCATGGAACTGTACCCTGCCCTGCTGGTGGAAAAACCTCGTCCAGATGCTATCTTTGGGGAGACCATGGTAGAGCTTGGAGCACCATTCTCCTTGAAAGGACTTATGGGAAATCCCATCTGTTCTCCTCAATACTGGAAGCCGAGCACCTTTGGAGGCGAAGTGGGTTTTAAGATCATCAATACTGCCTCAATTCAGTCTCTCATCTGCAATAATGTGAAGGGGTGTCCCTTCACTTCTTTCAATGTGCAAGATCCACAGCCTACCAAAACAGCCACCATCAATGCAAGTGCCTCCCACTCCAGACTAGATGACATTAACCCTACAGTACTAATCAAAAGGCGTTCAACTGAGCTGTAAAAGTCTACTGACCATATTTATTTATTTATGTGAAGGAATTTAATTTAATTATTTAATATTTATACTGAATTTTTTTTCATGTAACATCTTCCATAACAGAAGGCAATGTTCTTGAACAATGTTACATTTGTGAAGATTCCTCCGGTGTTTGTCCTTTAAATATGTGTTACCTGAAACTGAAAGGAAATCAGCATTCATTCCTCTACATAAGCCAGTGAGAAGGGAAATGAATTTTGATATCTTTATACTTGAATTTCAGATCATGAATTAGCTTAACAAGAACCAAGGAAAAATGTATGAATATGTGAGTGTTGTTACAAGATGAAAAATGCTGCAGGTATCAACACTGTTGGTTACACTGTGTCTTCTTTACCTATGATAGGAGCATGTAATGTGGAATTCGTCTTAAATCCTTGCATATCTTTATCTCATCAAACAAAGGGGTCCAAGTTCAGTTTTAAATAAGCATTTAAGGCAGATACTGACAACAATCTCATTTTTTTAAAATGTTGTCTTGAGACAAATAATTTGAAATTTCTAAATTGGGACGTTTGAATCACTTTTGAAAGCTCTTACTTTCTTAAGCTGTCAGGTTTGTACCGACATGGAGTAAACAGCTATCATAAACGTAAATCTCCAAAACTAGTAGAAATTATGTCATGATTGATGGTTAAGATACCATGTCAGGGATTGTCTTTTCTTAGAAGTAGTGAAAGCTACTTACTATGACAATCAGACCTTCCTTGTATGTCAAAATGCTGGTGTGGAAGGTGGTGGAGCCCGTGCTGCTCTGTCTTAACTATGAGTGTGAGCTTTAAAGCTCGTTGATGAGTGGTAGCCAGCAAAGCCTAGAGCAACAAAAGCGTTCTACAAAGGAACTAACCAAGAACAAAGAAGGGTTCCCAATTAAAGATCACATTCAGGGTTAAACTTCCAAAGGAGACATCCTGATCCTGGTTTTGTGCTGGCCTGGTACTCAGTAGGTTTTTGCTGTGAGGTTAAAGACTTGCCAGGGCTGAACTTCGAAACAGTTTTTCTGTTGCACAGTATGATGTAACAGTCCATCTCTCAATGCAATAGGTATCAGTGGCCTCGTGAGCTTCTTCACAATATTTGATATGTCTTCCAGCCCATTGAACCTGGACTGCAGAAGGCCCCATGTCATGTGTGAGCTCAGCCTGGATGCCAGCATTTGCTGCTCCTCTTAGTTCCGTTTCTCGTGGTCACTTTACTACGAGAAACGCTGATTGGGTTTTCGTAGCTGTGTTACCAGGTTTTTAGTATCAGAACTATTCTTTCTTTAACCTCTATTCATATTTTCTCTACTTGAAGTTTTACATTCAGGAAAACCTCAGCGCTCAGGACTACTATGTACCTCCCCTTTGGAGGGAAAAATTCATTTTTAGGTAAAAGGCAAAAATTTTTTAAAAATATTTTTTATTTATAATTATATGGAAGGGCCCTACCAAGATGCTAGAAATATAGGGAGTTCCTGACAAGAAATTTCCATTCTTATTCTGAAGAATTGCTTTCTTACTTAAAAACAAAGACAGTTTGTGAGTAGTTCTGGGCAATAGGGATAAATATAAAACAATAATGATGATCATTTTCTACATCTCATTATCAGCTGAGGTACTGTATATTACTGAATTTATTGAAGATAGTTATGTCTTTTAGACATTGTTGTTATAAACTATGTTTAAGCCTACTACAAGTGTTTCTTTTTTGCATTATGTTGGAATTGATGTACCTTTTTTATGATTACCTCTCTGAACTATGGTGTGAACAATCAAACAAAATGATGAGATTAACGTTCATGGATAAATTCTAAGAAAACTAGTGTATTTTTTTGAAAAGTTTGAAGTTAGAACTTAGGCTGTTGGAATTTACGCATAAAGCAGACTGCATAGGATCCAATATTGACTGACCCAAGCATGTTATAAAGACTGACATTTTAGACATTTTGAAGGCCCTGTAAGTGTTTATTAATTAGTTAGAACTTAATTGATTAAAAAATATATCCAAAGCACTATAGGCATTAGAATTC (SEQ. ID. NO.7) III. MOUSE COX-1AMINO ACID SEQUENCE MSRRSLSLWFPLLLLLLLPPTPSVLLADPGVPSPVNPCCYYPCQNQGVCVRFGLDNYQCDCTRTGYSGPNCTIPEIWTWLRNSLRPSPSFTHFLLTHGYWLWEFVNATFIREVLMRLVLTVRSNLIPSPPTYNSAHDYISWESFSNVSYYTRILPSVPKDCPTPMGTKGKKQLPDVQLLAQQLLLRREFIPAPQGTNILFAFFAQHFTHQFFKTSGKMGPGFTKALGHGVDLGHIYGDNLERQYHLRLFKDGKLKYQVLDGEVYPPSVEQASVLMRYPPGVPPERQMAVGQEVFGLLPGLMLFSTIWLREHNRVCDLLKEEHPTWDDEQLFQTTRLILIGETIKIVIEEYVQHLSGYFLQLKFDPELLFRAQFQYRNRIAMEFNHLYHWHPLMPNSFQVGSQEYSYEQFLFNTSMLVDYGVEALVDAFSRQRAGRIGGGRNFDYHVLHVAVDVIKESREMRLQPFNEYRKRFGLKPYTSFQELTGEKEMAAELEELYGDIDALEFYPGLLLEKCQPNSIFGESMIEMGAPFSLKGLLGNPICSPEYWKPSTFGGDVGFNLVNTASLKKLVCLNTKTCPYVSFRVPDYPGDDGSVLVRRST EL (SEQ. ID. NO.8)IV. MOUSE COX-2 AMINO ACID SEQUENCEMLFRAVLLCAALGLSQAANPCCSNPCQNRGECMSTGFDQYKCDCTRTGFYGENCTTPEFLTRIKLLLKPTPNTVHYILTHFKGVWNIVNNIPFLRSLTMKYVLTSRSYLIDSPPTYNVHYGYKSWEAFSNLSYYTRALPPVADDCPTPMGVKGNKELPDSKEVLEKVLLRREFIPDPQGSNMMFAFFAQHFTHQFFKTDHKRGPGFTRGLGHGVDLNHIYGETLDRQHKLRLFKDGKLKYQVIGGEVYPPTVKDTQVEMIYPPHIPENLQFAVGQEVFGLVPGLMMYATIWLREHNRVCDILKQEHPEWGDEQLFQTSRLILIGETIKIVIEDYVQHLSGYHFKLKFDPELLFNQQFQYQNRIASEFNTLYHWHPLLPDTFNIEDQEYSFKQFLYNNSILLEHGLTQFVESFTRQIAGRVAGGRNVPIAVQAVAKASIDQSREMKYQSLNEYRKRFSLKPYTSFEELTGEKEMAAELKALYSDIDVMELYPALLVEKPRPDAIFGETMVELGAPFSLKGLMGNPICSPQYWKPSTFGGEVGFKIINTASIQSLICNNVKGCPFTSFNVQDPQPTKTATINASASHSRLDDINPTVLIKRR STEL

What is claimed is:
 1. A vector for use in enhancing wound healingcomprising a promoter linked to a cyclooxygenase expression cassette. 2.The vector of claim 1, wherein the promoter is constitutively induced.3. The vector of claim 2, wherein the promoter is a cytomegaloviruspromoter.
 4. The vector of claim 1, wherein the cyclooxygenaseexpression cassette encodes a COX-2 gene product.
 5. A method forenhancing wound healing comprising the step of delivering the vector ofclaim 1 to the location of the wound; wherein the cyclooxygenaseexpression cassette is expressed, thereby enhancing wound healing. 6.The method of claim 5, wherein the vector is a adenoviral vector,adeno-associated viral vector, a recombination-defective retrovirus or aplasmid.
 7. The method of claim 5, wherein the vector is delivered byinjection, electroporation, or inhalation of an aerosol.
 8. The methodof claim 5, wherein the vector is in a saline solution, encapsulated inliposomes, in a polymer solution, in a gel, or lyophilized.
 9. Themethod of claim 5, wherein the promoter is cytomegalovirus.
 10. Themethod of claim 5, wherein the cyclooxygenase expression cassetteencodes for a COX-2 gene product.
 11. The method of claim 5, wherein thewound is a bone fracture or a skin wound.
 12. A method for enhancingwound healing following orthopaedic procedures comprising the step ofadministering the vector of claim 1 to the location of the procedure;wherein the cyclooxygenase expression cassette encodes for COX-2 geneproduct and wherein the cyclooxygenase expression cassette is expressedthereby enhancing wound healing.
 13. A method for treating pathologicalheterotopic ossification conditions comprising the steps of: identifyinglocations of heterotopic ossification in a patient with a heterotopicossification condition, and administering COX-2 selective NSAIDs to thepatient.
 14. The method of claim 13, wherein the heterotopicossification condition is fibrodysplasia ossificans progressiva, occursfollowing a hip replacement procedure or after an acetabular fracture.15. A method for inhibiting wound healing comprising the step ofadministering an effective amount of NSAIDs to the location of thewound, wherein the NSAIDs inhibit normal wound healing.
 16. The methodof claim 15, wherein the NSAIDs are COX-2 specific NSAIDs.
 17. A methodfor treating osteoporosis, OI and brittle bone conditions comprising thestep of administering the vector of claim 1, wherein expression of thevector enhances wound healing and bone formation.
 18. A composition foruse in wound healing comprising a formulated cyclooxygenase protein. 19.The composition of claim 18, further comprising a pharmaceuticallyacceptable carrier.
 20. The composition of claim 18, wherein thecyclooxygenase protein is COX-1, COX-2 or a combination of the two. 21.A method for treating wounds comprising the step administering thecomposition of claim 18 to a patient with a wound.
 22. The method ofclaim 21, wherein the composition is administered topically, orally,intravenously, nasally or locally.